a genetic linkage map of black raspberry (rubus ... · lated, saturated genetic linkage map of...

1 3

Theor Appl Genet (2015) 128:1631–1646DOI 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

its lack of improvement is low genetic diversity in elite cul-tivars and an untapped reservoir of genetic diversity from wild germplasm. In the Pacific Northwest, where most pro-duction is centered, the current standard commercial culti-var is highly susceptible to the aphid Amphorophora aga-thonica 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 resist-ance 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 link-age map using single-nucleotide polymorphism and trans-ferable (primarily simple sequence repeat) markers for F1 population ORUS 4305 consisting of 115 progeny that seg-regate for aphid resistance. Our linkage map of seven link-age groups representing the seven haploid chromosomes of black raspberry consists of 274 markers on the mater-nal 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 map-ping 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 earli-est 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;

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 [email protected]

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

http://crossmark.crossref.org/dialog/?doi=10.1007/s00122-015-2541-x&domain=pdf

http://dx.doi.org/10.1007/s00122-015-2541-x

1632 Theor Appl Genet (2015) 128:1631–1646

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Foolad et al. 1995; Hemmat et al. 1994; Rajapakse et al. 1995; Stockinger et al. 1996; Viruel et al. 1995). Advance-ments 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-Fernán-dez 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). Addi-tional 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 (Anta-naviciute 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; Wood-head et al. 2010), and an interspecific cross between dip-loid red raspberry and diploid black raspberry (Bushakra et al. 2012). While blackberry and red raspberry are highly heterozygous, black raspberry, particularly R. occidenta-lis, 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 Pomologi-cal 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 cul-tivars and only three black raspberry cultivars (‘Pequot’, ‘Niwot’, and ‘Explorer’). In addition, ‘Earlysweet,’ a selec-tion 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

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; Hell-man 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, inflam-mation, 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 (Anon-ymous 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 agath-onica 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 signifi-cantly increase the longevity of the plants, reduce insecti-cide use, and therefore improve profitability for the grower and quality of the environment.

A low level of genetic diversity in cultivated black rasp-berry has been found using molecular tools. Weber (2003), using RAPD markers in 16 black raspberry cultivars, deter-mined 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 rasp-berry 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 rasp-berry × black raspberry cross and found that the homozy-gosity 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 diver-sity. 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 frag-ments that identified 15 genotypes in the 22 samples col-lected. Dossett et al. (2012b) used SSR markers to examine

1633Theor Appl Genet (2015) 128:1631–1646

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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 (Dos-sett 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 ger-minated and evaluated for multiple traits including aphid resistance. From this study, three of 132 wild populations were determined to segregate for resistance to A. agath-onica. Two populations, ORUS 3817 collected from Maine, and ORUS 3778 collected from Ontario, Canada, were subsequently used to develop populations for genetic map-ping 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 resist-ance 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 fol-lowing 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, sus-ceptible 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

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 trans-ported 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 Bio-Tek, Norcross, GA, USA) was used as previously described (Gilmore et al. 2011).

DNA quantification and quality

Genomic DNA was quantified using Quant-iT™ Pico-green® 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

ORUS 4305

ORUS 3021-2

ORUS 4153-1Ag4 aphid resistance

‘Jewel’

NC84-10-3

‘Jewel’

ORUS 3778-1Wild, 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 Ameri-can large raspberry aphid that was crossed with aphid-susceptible ‘Jewel.’ One of the progeny from that cross, ORUS 4153-1 with pro-posed genotype Ag4ag4 representing the proposed gene conferring resistance, was used as the male parent and crossed with aphid-sus-ceptible ORUS 3021-2

1634 Theor Appl Genet (2015) 128:1631–1646

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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) fol-lowed immediately by a number (i.e., Ro11481, Ri13528) (Dossett et al. in press). All other markers are from pub-lished 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 gradi-ent 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 fluores-cent 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 pro-tocol began with an initial denaturation for 3 min at 94 °C followed by 10 cycles of 94 °C for 40 s, 65 °C (decreas-ing 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 for-ward primer, 0.5 μM WellRed labeled M13 primer (D2, D3 or D4), and 0.025 U GoTaq® Hot Start Polymerase (Pro-mega 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

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 elec-trophoresis. 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 Mas-ter Mix, 1 μM each forward and reverse primer. Each well was topped with one drop of mineral oil. The PCR ampli-fication 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 per-formed 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 bar-code adapters (Elshire et al. 2011). Annealed and quanti-tated unique barcode and common adapters were provided by the Buckler Lab for Maize Genetics and Diversity, Cor-nell 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

1635Theor Appl Genet (2015) 128:1631–1646

1 3

Tabl

e 1

Tra

nsfe

rabl

e lo

cus

prim

er s

eque

nces

use

d to

con

stru

ct th

e ge

netic

link

age

map

s fo

r bl

ack

rasp

berr

y F 1

pop

ulat

ion

OR

US

4305

Loc

usR

LG

3021

-2 a

llele

si

zes

4153

-1 a

llele

si

zes

Rep

eat m

otif

Forw

ard

prim

er s

eque

nce

Rev

erse

pri

mer

seq

uenc

eSo

urce

ER

ubL

R_S

Q07

-3_C

07_H

RM

_R

LG

77

NA

NA

NA

GG

AT

CA

AG

GA

GT

GA

GG

AT

GG

CC

GT

GG

TG

GT

TG

TTA

TG

TT

GB

usha

kra

et a

l. (2

012)

Ri_

1B16

_HR

M_R

LG

77

NA

NA

NA

CT

TG

GG

CA

GC

TT

TAG

CC

TT

TA

AG

AA

GA

AG

GG

TG

GG

TT

TC

A

Ru_

EE

2843

82_H

RM

_RL

G5

5N

AN

AN

AA

CG

GA

GG

AT

GA

CA

GA

GA

AC

CA

GG

TG

AG

GT

GG

GA

GA

TG

AT

G

Ro_

CB

Ea0

001L

24_S

SR2

275,

277

277,

279

(CC

A) 4

–(TA

) 7M

13-T

AA

AG

AA

AG

GG

GT

TG

TT

GC

GG

AC

GT

CT

CC

AT

TG

GG

AA

GA

AU

npub

lishe

d

Ro_

CB

Ea0

002P

01b_

SSR

122

6, 2

2822

8(T

C) 4

–(C

T) 7

M13

-CC

CT

CC

CT

CT

CT

CC

AG

TT

TC

GC

GC

TT

GA

GC

AT

CA

AA

TG

TA

Ro_

CB

Ea0

003K

17_S

SR1

307

307,

309

(TC

) 6M

13-G

GA

AA

GA

AA

AC

CC

TAG

CC

GA

CT

TAC

GC

TT

CT

TG

GC

TC

CA

C

Ro_

CB

Ea0

004G

23_S

SR4

509

507,

509

(AG

) 8M

13-G

AC

GC

GG

TG

AG

AT

TT

TG

AT

TG

TT

CC

CT

TT

GC

TT

TG

AG

AC

G

Ro_

CB

Ea0

009J

05_S

SR4

292

292,

294

(CA

) 4–(

GA

) 3M

13-C

CA

AG

TC

CA

AC

CA

CT

CA

CA

CT

TT

GC

TC

GT

CG

TAC

TC

AT

CG

RhM

003_

SSR

_RL

G3

322

7, 2

3623

6, 2

38(T

G) 1

0M

13-C

CA

TC

TC

CA

AT

TC

AG

TT

CT

TC

CA

GC

AG

AA

TC

GG

TT

CT

TAC

AA

GC

Cas

tillo

et a

l. (2

010)

RiM

017_

SSR

421

2, 2

1421

2(T

G) 6

M13

-GA

AA

CA

GG

TG

GA

AA

GA

AA

CC

TG

CA

TT

GT

GC

TTA

TG

AT

GG

TT

TC

G

Rh_

ME

a000

6bG

05_S

SR6

294,

303

294

(AA

G) 8

M13

-GA

AG

CA

GC

AG

CA

AG

AC

CT

TT

GT

TC

AG

GC

CA

GT

CA

AT

GT

CA

Cas

tro

et a

l. (2

013)

Rub

1C6_

SSR

624

4, 2

6026

2, 2

68(C

T) 1

5D

4‑T

CT

GC

CT

CT

GC

AT

TT

TA

CA

CA

GG

TT

TAG

GTA

AG

CA

AT

GG

GA

AA

GC

TC

Dos

sett

and

Finn

(2

010)

Ro_

CB

Ea0

010N

20_S

SR4

114,

118

118

(GA

) 9M

13-G

GG

GG

CT

TTA

CA

TC

AT

CA

TT

TT

CG

TAG

TC

TT

GC

CC

TT

GC

TD

osse

tt et

al.

(201

2b)

Ro_

CB

Ea0

011M

11_S

SR5

243,

245

241,

243

(AG

) 14

M13

-GG

GC

AT

GA

AC

CA

CA

TAA

AG

GT

CC

AT

TT

CC

AA

AA

CA

CA

TT

GA

Ri1

0139

_SSR

729

529

5, 3

14(T

C) 8

M13

-GT

CT

CG

GC

CG

AA

TAA

TAA

AC

AA

CA

CG

AA

GA

AC

AA

CG

AG

AG

AA

AA

Dos

sett

et a

l., in

pre

ss

Ri1

1086

_SSR

226

8, 3

0026

8, 3

02(T

C) 9

M13

-AA

AA

TT

CT

GA

TT

GG

GC

CG

AC

AC

AA

CA

CG

AA

GA

AC

AC

GA

GA

GA

Ri1

1795

_SSR

329

9, 3

1329

9, 3

13(G

AA

) 8M

13-A

TC

CA

AC

CC

TT

CA

TT

CT

CT

GT

TG

CG

AA

GA

CG

AG

GA

AG

AT

GA

AT

Ri1

2319

_SSR

330

429

2, 3

04(T

CA

) 7M

13-G

AG

TC

TG

ATA

TC

AA

GC

AG

CC

CT

AA

AG

GTA

GA

AG

TG

GA

GG

AC

TC

A

Ri1

3528

_SSR

746

0, 4

6346

3(T

C) 1

0M

13-C

TC

TG

CTA

CA

AC

CC

AA

CG

AG

TC

GG

CA

AT

TT

GG

AG

AT

TT

CT

TG

CT

Ri1

4075

_SSR

235

1, 3

5535

7, 3

59(T

C) 8

M13

-AC

AA

AT

TC

CA

GT

CA

GT

CC

AT

GC

CC

AG

AC

GC

AT

TAA

AT

CT

GT

CA

C

Ri1

6959

_SSR

728

3, 2

8628

6(T

C) 6

M13

-AA

AA

TG

TG

AT

TG

AG

CC

GA

CG

GG

GA

AA

AC

TG

AA

GA

AC

AC

GA

AG

Ri1

8886

_SSR

130

8, 3

1731

4, 3

17(A

TG

) 9M

13-C

CC

AA

AG

GA

CA

GA

AG

TAT

GG

AC

CG

GG

TC

TTA

CA

GG

CA

AG

TG

ATA

Ri2

0047

_SSR

537

6, 3

9337

6, 4

05(C

T) 9

M13

-CC

CT

GT

TT

GA

TC

TAT

TC

AA

TC

CC

GA

GG

AG

CA

GC

TT

GT

CG

AG

AT

Ri2

0466

_SSR

236

936

9, 3

74(G

A) 1

0M

13-G

GT

TT

TC

TG

GG

AA

AA

AC

AG

AA

GC

GC

GT

TT

TC

AC

TG

TT

CA

CT

TTA

Ri3

758_

SSR

239

2, 4

0640

0, 4

06(A

T) 7

M13

-GT

TT

TG

CC

TAC

GG

AC

TT

GA

AT

CT

CTA

TC

TC

TC

CG

TT

GT

GG

AT

TT

Ri5

037_

SSR

233

6, 3

3933

6, 3

39(G

AA

) 6M

13-C

AC

GA

GTA

AC

AC

TC

CC

AA

AT

GA

TC

TT

GG

AA

TT

GG

GG

TTA

TT

CT

G

Ro1

0488

_SSR

211

912

1, 1

31(T

C) 9

–(T

C) 1

4M

13-A

GG

GT

GC

GT

GT

CA

GA

AG

TAA

GT

GC

TG

ATA

GT

GG

GG

TT

TG

GA

TAA

Ro1

079_

SSR

422

3, 2

2522

3(T

A) 6

M13

-AA

AA

TG

GA

GA

CTA

GA

TC

CA

GC

GG

GC

AG

AG

AT

TT

GA

GG

TT

TC

TG

A

Ro1

1481

_SSR

615

615

6, 1

58(A

G) 6

–(TA

) 6–(

AT

) 5M

13-A

AG

ATA

AG

AG

GA

GA

AG

TG

CG

GA

CT

GT

TT

CC

AG

CA

AA

CC

TAA

CC

T

Ro1

2112

_SSR

615

7, 1

6714

0(T

C) 8

M13

-TA

CT

CC

CA

AA

AA

CC

CA

GA

AT

TG

GT

CT

GA

GC

AG

AA

AT

GG

GA

AA

TC

Ro1

4925

_SSR

711

711

4, 1

17(T

GT

) 7M

13-A

GC

TG

GT

CG

AA

GA

AG

GT

TTA

TG

AA

CT

TT

CT

CC

CG

TT

CT

CC

TAG

C

Ro1

5590

_SSR

617

9, 2

1120

1, 2

05(G

A) 6

M13

-GG

AG

CA

AG

AA

GC

CT

TG

AA

GA

TAG

TT

GC

CT

CT

GG

AT

TG

CT

TT

TAT

Ro1

6697

_SSR

415

2, 1

6016

0(A

T) 8

–(TA

) 6M

13-C

CA

GT

GA

GT

GA

GC

CT

TG

AG

ATA

AT

TT

GG

AA

GG

AA

TAC

GG

AA

CC

T

Ro1

682_

SSR

311

9, 1

2512

1(A

T) 6

M13

-AG

GA

GC

GA

TG

TTA

TAG

GC

AT

GT

TAG

AG

GG

AG

AA

AA

AG

GG

AG

TG

C

1636 Theor Appl Genet (2015) 128:1631–1646

1 3

Tabl

e 1

con

tinue

d

Loc

usR

LG

3021

-2 a

llele

si

zes

4153

-1 a

llele

si

zes

Rep

eat m

otif

Forw

ard

prim

er s

eque

nce

Rev

erse

pri

mer

seq

uenc

eSo

urce

Ro1

7045

_SSR

316

416

4, 1

67(T

GA

) 8M

13-T

CC

AA

CA

TT

GG

TG

AC

AG

TT

TT

CA

CT

TT

TG

CA

TC

TG

CT

TC

AT

CT

G

Ro1

7803

_SSR

213

9, 1

4713

9, 1

41(T

A) 1

0M

13-G

CC

CG

ATA

GA

TTA

AA

AG

GG

AA

AG

TT

CA

GA

AT

GC

AG

TT

GA

AA

CC

A

Ro1

8036

_SSR

110

4, 1

1911

9(C

CT

) 8M

13-C

TT

CT

TG

GG

AC

GA

AA

AA

CA

AA

CC

TG

TG

GA

TT

CA

GA

CG

AA

GA

TG

A

Ro1

9042

_SSR

620

119

6, 2

01(G

A) 6

M13

-GG

GTA

TAT

TC

CA

AA

AC

CC

CA

AT

TG

GG

TT

TC

AA

AG

GT

CA

AT

CT

CT

Ro2

0267

_SSR

415

915

3, 1

59(T

GA

) 8M

13-G

AA

CC

AA

AG

CT

TT

TG

AT

TG

GT

CG

TT

GG

AT

TT

CA

TG

GA

AA

GT

GT

C

Ro2

173_

SSR

419

9, 2

4020

3, 2

21(T

TA) 8

M13

-TA

TT

GG

GA

GT

GA

AA

GA

GC

CC

TAG

GT

GTA

TT

TTA

AT

GC

GG

TC

AC

A

Ro2

432_

SSR

511

4, 1

1611

4(T

C) 8

M13

-CG

GA

TG

AA

TT

TAA

GA

AA

GC

TG

GC

TT

CT

CA

AG

AA

CA

CG

GC

GA

T

Ro2

579_

SSR

418

117

9, 1

81(C

A) 1

0M

13-T

TT

TATA

TG

CT

TG

TC

CC

AC

AC

GA

TTA

TAG

AA

AT

TG

GG

CG

CA

CT

C

Ro2

827_

SSR

513

3, 1

4113

7, 1

41(C

T) 6

M13

-GC

GT

CT

GC

TT

TC

TT

CT

CA

GT

CT

GA

GC

GC

AG

AA

GC

AG

AC

TTA

TC

T

Ro3

003_

SSR

514

5, 1

9914

5, 1

52(G

A) 6

–(G

A) 7

M13

-AC

GT

TG

AT

CA

TAG

CC

TC

CA

AA

TC

TT

CC

CA

TAG

CA

AC

TC

TAT

CC

C

Ro3

017_

SSR

315

9, 1

7316

1(G

A) 7

M13

-CA

AC

CG

CT

TTA

AT

GA

AG

TG

TG

AG

CA

CA

AG

TAG

CA

CA

AC

TC

AA

CA

Ro3

237_

SSR

113

1, 1

3513

3, 1

35(T

A) 7

M13

-AA

CC

CA

AA

GC

TT

TC

CT

TC

TT

GT

AT

TG

GC

AG

GC

TT

TC

CT

TAC

ATA

Ro3

981_

SSR

611

5, 1

1711

5(T

G) 7

–(T

G) 7

M13

-GA

TC

TC

TG

AT

TC

CC

GC

AT

TAT

TA

AA

TG

TC

CT

TC

CT

GA

TG

AT

TG

G

Ro4

104_

SSR

718

1, 1

8518

1, 1

83(T

A) 7

M13

-AA

AG

CT

TC

CT

CA

TT

TT

GT

GA

GC

AT

GA

TAT

GA

CG

GC

TG

AG

AT

CA

A

Ro4

261_

SSR

420

4, 2

1920

4, 2

19(T

TC

) 9M

13-A

ATA

GC

AT

GG

AA

TC

CA

CT

CA

CC

TC

TC

AT

TC

CA

GA

TG

GG

TTA

TC

A

Ro4

345_

SSR

510

8, 1

1411

4(T

C) 7

M13

-TTA

CA

GC

AA

TT

GA

AG

GA

TG

AG

CA

AA

GA

AA

TAG

GG

AA

AG

GG

GG

AG

Ro4

532_

SSR

621

0, 2

1321

0, 2

13(T

TG

) 6M

13-A

GT

TC

AT

CA

AT

TT

GA

GG

GA

TG

GT

CG

AT

GA

TC

ATA

TC

AT

TC

CA

CC

Ro5

263_

SSR

620

1, 2

0320

1(G

A) 6

M13

-AA

CC

TT

TT

GC

GT

TT

GA

TAC

TC

CT

TT

GT

TT

GC

CT

TAG

AG

TC

CT

CC

Ro5

378_

SSR

420

719

1, 2

07(T

A) 8

M13

-TC

TT

CA

CA

CA

TG

TC

CA

CT

GG

TT

TC

AG

CT

GA

GT

TT

TT

GC

AG

AG

AT

Ro6

594_

SSR

117

1, 1

7717

1(T

TC

) 9M

13-T

TT

GA

GA

GG

AC

GA

AT

GT

CG

TTA

CT

GTA

ATA

CTA

GG

CT

CC

AC

CG

C

Ro7

270_

SSR

316

8, 1

7117

7(G

AA

) 8M

13-C

TC

AG

GA

AA

CC

GT

CA

TAC

TT

CC

TG

GT

CT

TC

CA

TAA

CC

CT

TC

AG

T

Ro8

167_

SSR

694

, 96

96(T

C) 6

M13

-CA

AT

TG

CA

CA

TAA

CC

CA

TC

AT

CG

AA

GG

AA

TG

CA

AA

AC

CA

GA

AA

G

Ro8

486_

SSR

217

2, 1

7817

2(C

T) 9

M13

-TC

GC

GC

TG

ATA

GT

GT

TT

CA

TAC

AA

GG

AA

TG

AA

ATA

GG

GA

CG

GT

T

Ro9

206_

SSR

513

5, 1

3912

6, 1

37(A

T) 8

M13

-AC

AG

TT

CC

TAC

AA

AG

GA

TC

GG

AC

AA

GA

TT

GT

CA

CG

TAC

TC

GG

AA

Ro9

324_

SSR

115

6, 1

6416

4, 2

01(A

G) 7

M13

-CC

TAC

TT

TC

AA

AG

CC

CA

TT

TT

GG

CA

AT

CA

CA

CA

TTA

AA

AG

GT

CC

Ro9

42_S

SR1

155,

161

161,

181

(GA

A) 7

M13

-AA

TC

GT

CG

CC

TG

CA

ATA

TT

TAC

CA

AA

TT

CG

AC

AC

CA

CC

TAT

CA

G

Rub

us11

0a_S

SR_R

LG

44

187,

207

203,

205

(TC

) 8M

13-A

AA

CA

AA

GG

ATA

AA

GT

GG

GA

AG

GT

GT

CA

GT

TG

GA

GG

GA

GA

AC

AG

raha

m e

t al.

(200

4)

Rub

us11

6a_S

SR_R

LG

44

222,

224

218,

224

(CT

) 12–

(T) 1

0M

13-C

CA

AC

CC

AA

AA

AC

CT

TC

AA

CG

TT

GT

GG

CA

TG

GC

CT

TT

TAT

Rub

us12

6b_S

SR_R

LG

22

171

157,

171

(CT

) 31–

(CA

) 22

M13

-CC

TG

CA

TT

TT

TC

TG

TAT

TT

TG

GT

CA

GT

TT

TC

TT

CC

CA

CG

GT

TA

Rub

us16

a_SS

R_R

LG

66

170,

172

164,

166

(AT

) 8(G

T) 1

1M

13-T

GT

TG

TAC

GT

GT

TG

GG

CT

TT

GG

GT

GT

TT

GC

CA

GT

TT

CA

GT

Rub

us22

3a_S

SR6

158,

162

158

(AT

)4–(

TA) 8

–(A

T) 1

0M

13-T

CT

CT

TG

CA

TG

TT

GA

GA

TT

CTA

TT

TTA

AG

GC

GT

CG

TG

GA

TAA

GG

Rub

us26

a_SS

R4

139,

141

137,

143

(CT

) 11–

(CA

) 29

M13

-AA

CA

CC

GG

CT

TC

TAA

GG

TC

TG

AT

CC

TG

GA

AA

GC

GA

TG

AA

A

Rub

us27

0a_S

SR_R

LG

33

182,

184

184,

186

(GA

) 10

D3‑

GC

AT

CA

GC

CA

TT

GA

AT

TT

CC

CC

CA

CC

TC

CA

TTA

CC

AA

CT

C

Rub

us27

5a_S

SR_R

LG

55

139,

165

165

(AG

) 27

M13

-CA

CA

AC

CA

GT

CC

CG

AG

AA

AT

CA

TT

TC

AT

CC

AA

AT

GC

AA

CC

1637Theor Appl Genet (2015) 128:1631–1646

1 3

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). Librar-ies 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 par-ent). The second sequencing run included 88 samples (26 black raspberry including parents, grandparents, stand-ards 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 pipe-line (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 Trim-momatic (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 Join-Map® v. 4.1 (Van Ooijen 2006). Loci were then organized into parental sets and subjected to the maximum likeli-hood (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 Ta

ble

1 c

ontin

ued

Loc

usR

LG

3021

-2 a

llele

si

zes

4153

-1 a

llele

si

zes

Rep

eat m

otif

Forw

ard

prim

er s

eque

nce

Rev

erse

pri

mer

seq

uenc

eSo

urce

Rh_

ME

a000

2cA

01_S

SR2

268,

274

268,

272

(CT

) 17

M13

-CC

CC

AA

AC

TC

CA

AA

AT

CT

CA

TT

CT

GC

TC

AT

CT

TT

GG

GG

TC

Lew

ers

et a

l. (2

008)

Rh_

ME

a000

7aB

01_S

SR_R

LG

55

148,

154

148,

152

(CT

) 15

M13

-TG

GT

GG

TT

CA

CC

GT

TC

AC

TAG

AA

AT

GC

TT

GA

AG

CC

GA

GA

G

Rh_

ME

a001

3bG

01_S

SR2

248,

250

248

(GA

) 38

M13

-CC

CT

CC

AT

CT

CC

AC

CA

TAA

AG

TAA

GG

CC

AC

CC

CA

TT

GA

G

Rh_

ME

a001

3cF0

8_SS

R1

254,

256

254,

266

(TC

) 15

M13

-TT

TG

TC

TC

CG

TC

TT

TT

TG

CC

CC

TC

CG

AA

GA

AA

AA

CA

GC

AG

ER

ubL

RSQ

_07-

4_D

05_S

SR_

RL

G6

626

026

0, 2

66(A

GC

) 7M

13-C

TT

CT

TT

CC

AA

CC

GA

TT

TC

AC

GA

AT

TG

AT

TT

CA

TC

AA

CC

Woo

dhea

d et

al.

(200

8)

Loc

us n

ames

pre

face

d w

ith R

o w

ere

deri

ved

from

bla

ck r

aspb

erry

(R

ubus

occ

iden

tali

s L

.), t

hose

pre

face

d w

ith R

i and

Rub

us w

ere

deri

ved

from

red

ras

pber

ry (

R. i

daeu

s L

.), t

hose

pre

face

d w

ith

Rh

wer

e de

rive

d fr

om b

lack

berr

y (R

ubus

sp.

). N

ames

in it

alic

s de

sign

ate

thos

e m

arke

rs th

at w

ere

used

to a

ncho

r th

e lin

kage

gro

ups

and

wer

e se

lect

ed b

ecau

se th

ey m

ap in

mul

tiple

Rub

us li

nk-

age

map

s. T

wo

mar

kers

(R

ub1C

6 an

d R

ubus

270a

) w

ere

desi

gned

with

a fl

uore

scen

t tag

on

thei

r fo

rwar

d pr

imer

. Eac

h en

try

incl

udes

the

linka

ge g

roup

to w

hich

the

locu

s m

appe

d, th

e al

lele

siz

e in

pop

ulat

ion

OR

US

4305

, the

rep

eat m

otif

, the

for

war

d an

d re

vers

e pr

imer

seq

uenc

e, a

nd s

ourc

e. T

he s

eque

nce

of th

e M

13 ta

g is

5′-T

GTA

AA

AC

GA

CG

GC

CA

GTA

GC

http://www.usadellab.org/cms/?page=trimmomatic

http://www.usadellab.org/cms/?page=trimmomatic

1638 Theor Appl Genet (2015) 128:1631–1646

1 3

ORUS 4153-1 using the regression algorithm of the map-ping 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 Micro-soft 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

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 popula-tion 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 prod-ucts 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 suc-cessfully, 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 (Supple-mentary Table 2).

GBS SNP markers

The first sequencing run of 95 samples generated 596 K sequence clusters/mm2 (optimal density is 750–850 K clus-ters/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 gener-ate an average number of reads per individual of 3,105,333, with 20,317,182 (5.8 %) of reads unaligned. Default TAS-SEL filtering parameters using the parent information iden-tified 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 differ-ences from the rest of the population and may be the result of a pollen contamination; however, the other four prog-eny 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

Table 2 Summary of loci mapped in F1 population ORUS 4305

Genotyping by sequencing (GBS) single nucleotide polymorphic (SNP) loci were generated by DNA digestion and subsequent high-throughput 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

Genotyping by sequencing (GBS)

Total number of GBS SNP identified over three sequencing runs

7911

Number of monomorphic or ambiguous loci 3472

Number of loci heterozygous in both parents 921

Number of loci heterozygous in ORUS 3021-2 318


Total scaffolds represented 356

Scaffolds mapping to multiple RLG 13

Total number of GBS SNP mapped 399

Simple sequence repeat (SSR)

Total number of loci screened 552


Number of loci that failed 118

Number of loci that are heterozygous in both parents 138



Number of loci mapped 70

High‑resolution melting (HRM)

Total number of loci screened 80


Number of loci that failed 0



Number of loci mapped 3

Anchor loci (26 SSR + 2 HRM) 28

Not mapped 16

Mapped 12

1639Theor Appl Genet (2015) 128:1631–1646

1 3

characteristics of which are summarized in Table 3. To con-struct the linkage map for ORUS 3021-2, five GBS-gener-ated SNP markers were removed for skewed segregation ratios, four were removed for creating double recombina-tion 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 centi-Morgans (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 dis-tance 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 GBS-generated SNP markers were removed for skewed segrega-tion ratios, 14 were removed for creating double recombi-nation 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 dis-tance 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 con-struction, 230 (92 %) segregated as expected, either 1:1 or 1:2:1; a single locus (0.4 %) varied from expected at a sig-nificance 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 popu-lation. 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

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 mark-ers 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 mark-ers 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 identi-fied 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 link-age 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 devel-oped 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 transfer-able 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

1640 Theor Appl Genet (2015) 128:1631–1646

1 3

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 Ta

ble

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3

S47_4283150.0S83_319151.0

S487_12006612.2S144_11643115.8S24_34854719.5S671_6274822.5S64_16566624.6S28_10718625.6S87_64177S252_23248928.7S114_4071429.7S380_6288830.7S316_18601739.5S431_3718740.7S842_37425S41_4359946.8S371_4918051.1S376_162362Ro11481_S32855.1S215_21829559.3

SQ07-4_D05_RLG6_S1469.9S818_2198170.2S477_11743674.6S1797_55677.4S447_6162678.9S394_12494684.4S977_2603487.5S99_10480091.8S122_74787S187_259241S140_40577

93.9

S196_140013105.1S684_63660108.5Ag4_AphidRS99_32802110.5S525_75530111.4S275_97574S474_45156112.4S79_351594114.3S65_461926121.7

S33_584984130.9S457_17312132.9

S214_34656142.0Ro4532_S13145.8S13_940743S63_573175149.4

ORUS 4153-1RLG6

Fig. 2 Rubus linkage group (RLG) 6 for black raspberry map-ping 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)

1641Theor Appl Genet (2015) 128:1631–1646

1 3

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 Arabi-dopsis 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

S20_7365640.0S70_1502280.1S335_263801.1

S20_1278767.7S42_2371478.9Ro_CBE2P01b_S7110.0

Ri18886_S915.2

S134_21773418.0

S3_79284826.2

S75_38103033.3

S15_27431636.6S81_16831038.3

S97_27243243.3S154_19064843.8S234_25823145.0Ro942_S23446.1

S88_9301454.0S10_61726958.4S804_5510660.0S565_2093062.1Rh_MEa13cF08_S8262.7Ro_CBEa3K17_S56564.7S143_18123165.5S648_1874868.8Ro9324_S28969.7S929_43718Ro6594_S67470.3Ro18036_S32270.4S14_95252571.2S414_7874S426_20245S142_215410S798_31088S999_22487

71.3

S721_5921471.7Ro3237_S19972.6S921_1975673.6S278_21818475.6S472_8982577.5

Consensus_RLG1

S2_6508880.0Ri37580.9S2_7395701.0

S71_4209054.5S7_3795065.3S7_2955706.2

Ri3758_S215.3S4_140544015.7S2_37231916.9S17_20792819.8S4_72597825.2S4_14258425.5S62_439154S14_87796028.3S14_4408132.2S468_9215032.4Ro10488_S42033.2S32_37570434.4S124_71435.2S22_49375835.8Ri14075_S3641.4S295_21736943.5S418_9149643.8S77_52263543.9S294_23396644.8S152_9416645.6S615_6618146.3S77_15093946.4S294_240516S269_6575747.4S112_1092748.5S217_52295Ro_CBEa1L24_S38248.8S456_103577S332_2523849.0Ri1108650.5Rh_MEa2cA01_S75551.1Ro8486_S33052.4S134_8395553.4S419_74798S134_74067S241_119603

53.5

S727_3015254.6Rh_MEa13bG01_S32455.9Ro17803_S17657.1S176_29510158.4S348_11401S650_32526S458_3294S714_73912

59.3

S590_9650S449_31741S169_39974

60.3

S149_32222662.0S353_15601463.4S23_12019964.6S10_6681370.4S53_41925370.7Rub126b_RLG2_S62270.8

Consensus_RLG2

S107_1242430.0S46_5798900.9S29_1687572.4S73_337544.3S46_7026366.4S139_1447318.7S142_7482S95_4156010.2Ro3017_S33410.3S139_16286210.7S75_8140612.4S29_64781512.5Ro7270_S959S6_27205613.4S177_26920913.5S107_11841113.9S113_30889518.1S40_48829218.8S27_90319619.8S113_30504121.3S111_34960223.1S80_303146Ro1682_S22624.6S239_14007324.9S233_65965S45_7146325.7S202_20308927.8S226_28513328.4S80_15191128.6S355_3031831.5S279_9615431.6S194_2793332.4S409_7255232.5S318_15570732.7S409_7263033.3S86_27267634.7S408_15655336.1S115_21194741.6S52_42929741.8S261_15379842.2S231_2943044.2S35_19071445.9RhM003_RLG346.3S168_5597050.0S380_13859550.1S76_22356951.6S167_27181054.1S25_88629556.5S167_30558357.5S175_12066958.4S201_5009158.5S156_26319559.3S209_270566S156_14740959.5S209_26570659.8S118_11362660.6S21_89707363.3S21_21318965.0S31_31468267.0S26_420968.0S314_17912568.2Ro17045_S2669.7S26_33582770.3Ri12319_S7270.7Rub270a_RLG3_S7272.0S119_192278S68_47734073.2

Consensus_RLG3

Rub116a_RLG4_S4620.0S1_23971010.9Ro16697_S1013.1RiM017_RLG4_S1013.5S101_2785504.2S462_772585.4S1_18831747.7

S1_4248710.8S1_864113.8S153_12403213.9Rub26a_RLG4_S814.3Ro4261_S114.7

Ro_CBEa9J05_S822.8S391_17396525.3S37_47982928.2S440_12312132.0S288_5268933.5Ro_CBEa4G23_S5433.9S357_17760435.9S357_17755036.4S485_4116037.8S327_60426S30_58209339.2S258_21261940.0Ro2579_S228Ro20267_S22841.1

Ro2173_S10844.4S174_26958945.5S109_30746351.6S1023_1606953.4S364_8065753.6S537_588153.7S116_11801954.0S502_12638555.0S537_5882S630_40278S314_148478

55.3

S529_2762955.7S10_124921157.2S850_1450257.5S161_35531257.7S469_6114058.3S235_14768960.0S494_1284961.2S1174_1918662.2S653_2131863.2Ro_CBEa10N20_S10363.9Ro5378_S40064.0S103_169793S398_55678S494_12860

64.2

S225_13886965.2S454_13254865.3S454_5575866.2S253_2239669.3Rub110a_RLG4_S10069.9S612_66375S723_48232S120_78654

70.3

S120_19990371.2S147_37541071.6Ro1079_S12073.8S509_8496474.6

Consensus_RLG4

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 rasp-berry aphid is shown in blue bold font (color figure online)

1642 Theor Appl Genet (2015) 128:1631–1646

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S26_2105800.0

Ro2827_S122.0S12_4095153.1RuEE284382_RLG5_S1786.8S11_5661997.5S11_7017917.6Ro_CBEa11M11_S512.0S5_48146514.7S5_67204815.6S386_16289217.8S247_26381018.5Ro9206_S13820.0S247_26443920.7S138_17269120.8Rh_MEa7aB01_RLG5_S9622.8S96_1053924.0S222_441524.9S190_16589825.9S134_33365526.9S85_20933627.3S146_17848328.2S78_38674229.2S134_39580829.8S51_580471S240_27459330.5S90_26013531.5S38_22691332.6S90_1694433.7Ri20047_S9033.9Ro4345_S107936.0S39_5002641.2S259_4618142.9S427_12160444.2S220_21631844.5S259_10091344.8S220_21634845.0S733_2764345.9S344_1249247.0S127_26572448.8Rub275_RLG550.5S530_10698351.5S1127_1604052.1S411_144987S1313_172353.2S276_21988953.6S395_5940754.3Ro3003_S116354.8S654_4226155.4S776_3374756.0S536_10718756.8S323_2281957.3S117_46256058.2S505_6131258.6S102_135460S961_1472363.6Ro243264.6S402_6144166.4S171_21363867.4S93_653069.2S1029_1216969.7

S59_19004474.6S387_822975.9S548_8920776.3

S370_4540479.1

Consensus_RLG5

S47_4283150.0S83_319151.2

Rub16a_RLG6_S5711.6S487_12006612.0S47_29399313.5S144_11643115.8S155_36862317.7Ro15590_S4818.7Ro3981_S7818.8S24_34854719.5S671_6274822.6S64_16566624.9S28_10718625.8S28_6454226.0S87_25364928.0Ro8167_S2828.9S252_23248929.1S87_6417729.2Ro12112_S11430.0S114_4071430.2S380_6288830.6

S215_21829538.4S316_18601739.0S842_3742540.2S41_4359940.4S371_4918040.8Ro11481_S328S376_16236241.1S477_11743641.5SQ07-4_D05_RLG6_S1442.0S818_2198142.8S447_6162643.3S1797_55643.7S977_2603444.5S394_12494645.2S122_74787S140_4057746.7S99_10480047.4S187_25924148.6S99_32802Ag4_AphidR50.0S780_4708350.1S256_6894750.3S266_8373750.4Ro5263_S26650.8S525_7553051.2S474_45156S275_9757451.8S79_35159453.8S238_3352656.5S492_5694958.1S1158_232860.9S122_23044561.9Rub1C6_S9964.0S474_1989865.0S260_422865.1S165_31550067.7S136_34690468.2S331_12082069.9S33_58498470.4S49_53498874.9S457_1731276.4Rub223a_S17979.9S214_3466380.7Rh_MEa6bG05_S1381.7S214_3465685.6Ro4532_S1386.5S13_940743S63_57317590.2

Consensus_RLG6

S1089_192380.0

Ri135286.3S44_5999596.8

Ro14925_S27911.4Ro4104_S42112.7

S151_13483617.2S151_3494618.3RiCHEa1B16_RLG7_S57419.5S325_10908020.8S121_41027222.0S219_19814422.3S219_19482522.6S249_5242423.0S604_6657724.8S547_5250325.5S1034_2602225.9S277_21134626.1S681_5839126.9S142_750827.1S781_396427.2S399_120712 S609_5457027.3S161_75403 S500_53440S608_26380 S799_5567227.4S1001_1949027.6S595_8436027.7S496_4420827.8S212_266279 S1104_111328.0S500_4574928.2S495_85717 S756_6580128.4S475_1639528.6S337_4227428.8S337_12647629.0S244_154037 S471_11455529.2S651_6131930.2S464_4128230.4S341_2492031.2Ri10139_S56632.2S577_1016032.4S780_975432.6S1391_623133.5Ri1695934.0S287_11218834.9S473_1676737.1S329_8203337.2S563_1506137.4S540_8406138.4S1212_14404 S384_4975239.4S472_8989140.1S61_29520840.4S705_6439141.0S115_45212 S110_26354441.6S61_4079542.3S865_348042.5SQ07-3_C07_RLG7_S14842.7S110_36217043.4S200_23779244.4S232_13449044.6S251_1708447.5S200_24267148.8S272_15858149.4S94_31348850.0S173_27814351.8S272_7119752.9S43_65681553.6S631_7869160.2S129_15348864.2S98_27461665.7S60_60405068.7

S34_75915881.0

Consensus_RLG7

Fig. 3 continued

1643Theor Appl Genet (2015) 128:1631–1646

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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 span-ning 376.6 cM, respectively (Ward et al. 2013). While diges-tion by a more frequent restriction enzyme cutter for GBS may be a way to increase the number of SNP loci identi-fied, 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 assem-bly; 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 ‘Baum-forth’s A’ and confers race-specific resistance to three biotypes of the European large raspberry aphid, Ampho-rophora 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 fine-mapping efforts and comparative mapping to red raspberry. This linkage map region is associated with many quantita-tive 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 trans-ferable markers. The presence of SSR and HRM markers selected from the literature, along with the other transfer-able 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 popu-lated 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 Coordina-tor performed marker screening, selected anchor markers, ran and scored all markers, constructed the genetic linkage map, and wrote the manuscript. DWB developed a cus-tom pipeline for bioinformatic analyses, and performed GBS SNP calling. MD developed the mapping popula-tion, 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

Table 4 Summary of the genomic scaffolds with loci on more than one Rubus linkage group (RLG)

Each entry details the linkage group and the parental map on which the loci are found and the type of locus, either single-nucleotide poly-morphic (SNP) or simple sequence repeat (SSR)

Scaffold RLG Parent SNP, SSR

S10 1, 2, 4 4153-1 3, 0

S14 1, 2 3021-2 2, 0

S14 2, 6 4153-1 1, 1

S21 3, 7 3021-2 2, 0

S26 3, 5, 7 3021-2 3, 0

S71 1, 2 3021-2 1, 1

S78 5, 6 3021-2 1, 1

S115 3, 7 3021-2 2, 0

S134 2, 5 3021-2 2, 0

S134 1, 2, 5 4153-1 3, 0

S142 1, 3 3021-2 2, 0

S142 3, 7 4153-1 2, 0

S161 7 3021-2 1, 0

S161 4 4153-1 1, 0

S279 3, 7 4153-1 1, 1

S380 3 3021-2 1, 0

S380 6 4153-1 1, 0

S472 1, 7 3021-2 2, 0

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calling and other bioinformatic analyses, BLAST analyses, and linkage mapping. BSG assisted in developing and per-formed 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 fund-ing) and contributed computational resources and bioin-formatics analysis. CEF PI on NIFA SCRI grant (project main funding), helped assemble and phenotype the germ-plasm, 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 Lew-ers (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 (Univer-sity of Florida, Gainesville, FL, USA) and two anonymous review-ers for their helpful suggestions. The authors thank the Washington Red Raspberry Commission, the Oregon Raspberry and Blackberry Commission, the North American Raspberry and Blackberry Grow-ers 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 Agri-culture (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 infor-mation 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 Crea-tive Commons Attribution 4.0 International License (http://creativecom-mons.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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