Two CAPS markers predict Verticillium wilt resistancein wild Solanum species

Pedro Uribe • Shelley Jansky • Dennis Halterman

Received: 12 December 2012 / Accepted: 23 September 2013 / Published online: 6 October 2013

� Springer Science+Business Media Dordrecht (outside the USA) 2013

Abstract Verticillium wilt of potato is a persistent

problem in the USA and worldwide. The disease,

which is caused primarily by the fungus Verticillium

dahliae, is difficult to manage, causes yield losses, and

contaminates soil for subsequent plantings. Control

strategies based on host resistance are seen as long-

term, stable solutions, but difficult to achieve given the

genetic nature of the host and the challenges associ-

ated with resistance evaluations. To provide breeders

with marker-assisted selection opportunities, we gen-

erated a pair of cleaved amplified polymorphic

sequence molecular markers within the coding region

of Ve2, a potato gene with homology to the tomato Ve1

gene that confers resistance to V. dahliae. The position

of the marker was determined according to the

consensus sequences of Ve2 homologs of wild Sola-

num species with resistance to V. dahliae. Marker

testing indicated their broad applicability, being able

to track the resistance to V. dahliae in progeny

containing genetic information derived from species S.

chacoense, S. brevicaule, S. berthaultii, S. tarijense,

and S. tuberosum. Furthermore, the two isolates of V.

dahliae used in our inoculation experiments differed

in virulence and demonstrated specificity for some

wild potato species. Experimentation leading to the

development of the markers and tests of their useful-

ness against a wide range of diploid potato germplasm

is presented.

Keywords Verticillium wilt � Potato early

dying � Molecular marker � Disease resistance

Introduction

Verticillium wilt (VW) of potato is a widespread,

persistent problem across virtually all the production

areas in the USA (Rowe et al. 1987; Rowe and

Powelson 2002; Omer et al. 2008). The disease is

mainly caused by the soil-borne fungus Verticillium

dahliae (Rowe et al. 1987; Rowe and Powelson 2002).

Verticillium can survive on infected debris, in the soil

and on tubers, and can be carried into other production

areas on contaminated soil or on infected material

(Frost et al. 2007).

V. dahliae is an imperfect fungus that produces two

types of asexual structures, conidia and microsclero-

tia. Conidia are usually found inside the colonized

xylem of the plants, while the microsclerotia are

P. Uribe

Department of Plant Pathology, University of Wisconsin-

Madison, Madison, WI 53706, USA

S. Jansky

Department of Horticulture, University of Wisconsin-

Madison, Madison, WI 53706, USA

S. Jansky � D. Halterman (&)

Vegetable Crops Research Unit, U.S. Department of

Agriculture, Agricultural Research Service, Madison,

WI 53706, USA

e-mail: dennis.halterman@ars.usda.gov

123

Mol Breeding (2014) 33:465–476

DOI 10.1007/s11032-013-9965-2

typically long-term resting structures found in the

cortex of colonized, senescing stems (Schnathorst

1981; Perry and Evert 1983; Frost et al. 2007).

Microsclerotia develop on senescing tissue and serve

as an overwintering mechanism for the fungus to

initiate a new disease cycle the following year

(Schnathorst 1981; Perry and Evert 1983).

Current VW control strategies rely on the use of

expensive and environmentally harmful soil fumigants

such as metam sodium or methyl bromide. Crop

rotation as an alternative for reducing the presence of

inoculum in the soil is seen as a useful measure

although the long-term survival of microsclerotia

tends to hamper its effectiveness. Biological control

using wet manure as a soil amendment in pre-plant

operations has been reported as an option for control-

ling the pathogen (Powelson and Rowe 1993),

although its full utility has not been tested. Breeding

for disease resistance against this fungus is another

method that is employed to control the disease and, in

light of the limited success of the above-mentioned

management methods, it represents the best long-term

effective control strategy currently available (Powel-

son and Rowe 1993; Jansky and Rouse 2000; Jansky

2009; Bae et al. 2009).

Resistance against V. dahliae has been identified in

wild potato species (Lynch et al. 1997; Jansky and

Rouse 2000; Concibido et al. 1994; Jansky 2009; Bae

et al. 2009). For example, in S. chacoense a single gene

was found conferring resistance to the closely related

pathogen V. albo-atrum (Lynch et al. 1997). Jansky

and Rouse (2000) developed two diploid interspecific

hybrid clones (C287 and C545) with resistance to V.

dahliae conferred by complementary gene action. In

tomato, resistance to this pathogen is controlled by a

single dominant gene called Ve (Diwan et al. 1999;

Kawchuk et al. 2001). Two copies of Ve (named Ve1

and Ve2) are present within the tomato genome

(Diwan et al. 1999; Kawchuk et al. 2001; Fradin

et al. 2009) but only Ve1 appears to be functional

(Fradin et al. 2009, 2011). Ve encodes a membrane

receptor protein containing leucine-rich repeats with a

putative function of recognition of pathogen elicitors

(Kawchuk et al. 2001; Fradin et al. 2009; de Jonge

et al. 2012). Prior investigation of the function and

conservation of resistance pathways between tomato

and potato indicated that either of the tomato Ve1 or

Ve2 genes could confer resistance to V. dahliae, a

result that indicates a conservation of VW resistance

signaling mechanisms between both species. This

raised the possibility that orthologous Ve genes are

present in some resistant potato lines and furthermore

that orthologs of Ve1 or Ve2 in potato are functional.

Simko et al. (2004) searched for Ve orthologs in

tetraploid potato and found high sequence identity to

both Ve1 and Ve2 in the resistant cultivar Reddale

(83–90 and 74–91 % respectively).

Commercial potatoes are tetraploid, heterozygous

clones that require a long time to breed (up to

15 years). Given their heterozygotic nature, efforts

to create new populations containing improved dis-

ease resistance traits are slow to yield acceptable

varieties. Nonetheless, some commercial varieties

such as Ranger Russet and Bannock Russet exhibit

intermediate resistance to V. dahliae while others such

as Russet Burbank or Russet Norkotah are susceptible

(Atallah et al. 2007). Because crossing polyploid

populations and selecting promising progeny is a

lengthy, difficult process, the use of markers linked to

traits of interest can speed the selection process

considerably. Single nucleotide polymorphisms

(SNPs), short sequence repeats (SSRs), random frag-

ment length polymorphisms (RFLPs) and cleaved

amplified polymorphic sequences (CAPS) are some of

the types of genetic markers used in breeding

purposes. With this in mind, Simko et al. (2004) was

able to find a marker linked to the potato Ve1 (StVe1)

locus, but the sequence variation observed was linked

to the susceptible allele. Bae et al. (2008) developed a

CAPS marker based on the orthologous sequence of

Ve2, using the diploid interspecific clones C287 and

C545. A good correlation was found between popu-

lations derived from this cross and resistance to the

pathogen.

We sought to develop a molecular marker for V.

dahliae resistance in potato using polymorphisms

present in wild Solanum populations. The usefulness

of marker-assisted selection for VW resistance would be

improved if the marker system were effective for a broad

range of wild Solanum species. Species and populations

for marker development were selected based on previ-

ous resistance screening data available in the US Potato

Genebank (NRSP6). Based on this information, a subset

of wild Solanum species with documented resistance

and susceptibility to V. dahliae was selected for testing.

Here we present the results of evaluations aimed at

determining the presence or absence of resistance to V.

dahliae in wild Solanum populations. We present data

466 Mol Breeding (2014) 33:465–476

123

showing the presence of polymorphic regions within

Solanum Ve2 homologs and the development of new

molecular markers for V. dahliae resistance screening

that are more broadly applicable than previously

published markers due to their ability to track resistance

from multiple genetic sources.

Materials and methods

Plant material

Botanical seed from wild Solanum populations was

obtained from the USDA Potato Genebank in Stur-

geon Bay, WI, USA. Populations were selected from

the Germplasm Resources Information Network

(GRIN) database based on consistent resistant or

susceptible responses in previous challenges with V.

dahliae. A total of 11 putatively resistant accessions

from 10 Solanum species and five putatively suscep-

tible accessions from four Solanum species were

included in this study (Table 1).

Plant growth and inoculation

Botanical seed was treated for 24 h in gibberellic acid

(1,500 ppm) to overcome dormancy, and approxi-

mately 100 seeds per accession were planted in

15 9 10 cm plastic pots filled with soil-less potting

mix (Metromix 360, SunGro Horticulture, Bellevue,

WA, USA) and covered with a thin layer of vermic-

ulite. Seedlings were grown in a greenhouse with 18 h

of daylight, a daytime temperature of 22 �C and a

night-time temperature of 18 �C.

V. dahliae inoculum was produced according to the

method of Bae et al. (2011). Briefly, 2-week old,

Table 1 Verticillium dahliae disease resistance screening of wild Solanum populations

Population name ID E10 Avga V18 Avga Avgb,d,e NRSP6 ratingc

S. brevicaule BRV 246536 0.0 ± 0.0 0.4 ± 0.5 0.22a Res

S. microdontum MCD 265881 0.6 ± 0.0 0.8 ± 0.4 0.68ab Res

S. chacoense sb. chacoense CHC 133619 0.8 ± 0.1 0.6 ± 0.3 0.70ab Res

S. berthaultii BER 310981 1.2 ± 0.5 0.4 ± 0.2 0.75abc* Res

S. bulbocastanum BLB 275193 1.4 ± 0.6 0.9 ± 0.4 1.16abcd Res

S. infudibuliforme IFD 320295 1.4 ± 0.0 1.3 ± 0.7 1.33abcd Res

S. kurtzianum KTZ 442680 1.9 ± 0.5 0.9 ± 0.2 1.38abcd* Res

S. brevicaule BRV 472974 2.1 ± 0.2 1.3 ± 0.8 1.69abcd Res

S. boliviense BLV 310974 2.0 ± 0.4 1.4 ± 0.0 1.70abcd* Sus

S. bulbocastanum BLB 255518 2.2 ± 0.6 1.3 ± 0.4 1.71abcd Sus

S. stoloniferum STO 186545 1.6 ± 0.4 1.8 ± 0.3 1.73abcd Res

S. boliviense BLV 265578 2.6 ± 0.6 1.1 ± 0.7 1.84abcd* Res

S. stoloniferum STO 255533 1.9 ± 0.5 2.3 ± 0.6 2.07bcd Sus

S. verrucosum VER 275259 2.6 ± 0.3 2.1 ± 0.5 2.33bcd Sus

S. candolleanum CND 265865 2.5 ± 0.7 2.3 ± 1.4 2.37bcd Sus

S. verrucosum VER 195170 2.8 ± 0.3 2.1 ± 1.4 2.44 cd Res

S. candolleanum CND 210055 3.5 ± 0.3 1.7 ± 1.5 2.56d Res

a Data presented are the average of two independent trials ± standard deviation, normalized to the ratings of the mock-inoculated

plants. The original rating scale of 1–4 of Bae et al. (2011) was therefore modified to reflect this. Thus: 4 = a dead or almost dead

plant; 3 = severe chlorosis, necrosis; 2 = chlorosis with less than 10 % necrosis; 1 = cotyledon chlorosis; 0 = healthy plantb Overall rating for population was calculated as the average of the ratings of the independent disease screens for both pathogen

isolatesc NRSP6 (National Repository Special Program 6) located in Sturgeon Bay (WI, USA) contains voucher information for the species

testedd The letters next to the data indicate not statistical difference in Tukey’s HDS tests in the pathogenicity to each isolatee The number of ‘‘*’’ next to the data indicates the significance according to t tests of the difference in the pathogenicity of E10 and

V18 on each accession. * p \ 0.1

Mol Breeding (2014) 33:465–476 467

123

actively growing mycelial plugs of V. dahliae grown

on agar plates were used to inoculate 200 ml of

Czapek-dox broth. The inoculum was shaken at 25 �C

for 10 days in the dark.

Four-week-old seedlings from each of the acces-

sions tested were inoculated with a spore suspension

of V. dahliae (isolate V18, obtained from an infected

Wisconsin field-grown potato plant) normalized to a

concentration of 5 9 105 conidia/ml, quantified with a

hemacytometer. Seedlings were gently removed from

the potting mix and their roots were rinsed in tap water

and then dipped in 250 ml of the inoculum suspension

for 30 min. Subsequently, 42 seedlings per accession

were transplanted into 48-well plastic flats containing

soil-less potting mix. Six control seedlings were

similarly treated with sterile water as a control and

placed into the remaining six wells in the flat; while

repotting the seedlings, care was taken to minimize the

possibility of cross contamination. The flats were

placed in a greenhouse with 18 h of daylight, an

average temperature of 30 �C during the day and

26 �C during the night. The flats were watered as

needed.

Plants were scored for disease symptoms every

5 days for 4 weeks, starting on the eighth day after

treatment. In addition, on the third day after inocula-

tion an initial baseline score (i.e. an initial disease

score aiming to record the effect that the dipping and

repotting had on the seedling) was taken and

subsequent score ratings were compared to this value.

The rating system consisted of a 1–5 scale where

1 = a dead or almost dead plant; 2 = severe chloro-

sis, necrosis; 3 = chlorosis with less than 10 %

necrosis; 4 = cotyledon chlorosis; 5 = healthy plant

(Bae et al. 2011).

Since the accessions were heterogeneous popula-

tions of wild Solanum species, we expected to observe

variability in developmental rates, germination and

disease resistance within each population. Therefore,

individual seedlings were scored for developmental

stage of growth during the resistance screening period.

A scale of 1–5 was used to evaluate the developmental

stage of the seedlings 3 days after treatment and on

each disease scoring date. The rating system used was:

1 = cotyledons opened; 2 = first true leaf opened;

3 = second true leaf opened; 4 = third true leaf

opened; 5 = beyond stage 4.

Disease score data were expressed as the difference

in disease symptoms between the non-inoculated and

inoculated seedlings. To investigate the possibility of

developmental stage and disease severity interactions,

correlation tests between the developmental stage of

the seedlings and their corresponding disease severity

were done. To gather more information on the

resistance or susceptibility of the wild Solanum

populations to V. dahliae, the same sets of experiments

were repeated with a second strain of the pathogen

(isolate E10, also isolated from a Wisconsin field-

grown potato plant). Disease resistance experiments

were repeated twice for each isolate. Independent

sample t tests were used to compare the effects of

isolate choice on the mean infection ratings of the

accessions. Tukey HSD analysis was done to statis-

tically determine which species were more resistant to

a particular isolate, and analysis of variance was used

to determine if any isolate was more pathogenic than

another.

DNA extraction

Plants screened for symptoms were grouped by

resistance or susceptibility phenotypes. DNA was

obtained from a total of 12 plants (six plants of each

group) for each of the potato accessions tested. Total

genomic DNA was extracted using a DNeasy kit

(Qiagen, Valencia, CA, USA).

PCR amplification of Ve2 homologs

Genomic DNA from each of the accessions tested

for resistance or susceptibility to V. dahliae

(Table 1) was used as a template to amplify a 1.6-

kb region orthologous to the tomato Ve2 gene using

the primers C287F1 and C287R1 (Bae et al. 2008).

Fifty-microlitre reactions were prepared using 1 ll

of genomic DNA as template (25 ng), 45 ll Plati-

num PCR Supermix High Fidelity (Invitrogen,

Carlsbad, CA, USA) and 2 ll each of primers

C287F1 and C287R1 (800 nM). PCR amplification

conditions comprised a 94 �C denaturing step for

2 min, followed by 40 cycles of 94 �C for 30 s,

50 �C for 40 s, and 68 �C for 2 min, with a final

extension at 68 �C for 15 min.

Cloning and sequencing of Ve2 homologs

PCR products were visualized after electrophoresis

on 1 % agarose gels stained with ethidium bromide.

468 Mol Breeding (2014) 33:465–476

123

Bands of the correct size (1.6 kb) were excised and the

PCR product cleaned using a MoBio UltraClean 15

Gel isolation kit (MoBio, Carlsbad, CA, USA) using

the manufacturer’s instructions. PCR products were

ligated into pGEM-T Easy (Promega, Madison, WI,

USA) according to the manufacturer’s instructions.

Escherichia coli competent cells were transformed

with ligation products using a heat shock protocol.

Sequencing reactions were done at the University of

Wisconsin Biotechnology Center using primers SP6

and T7. Sequence analysis was performed using the

Lasergene software suite (DNAstar, Inc., Madison,

WI, USA).

Marker design and testing

Sequence alignment of Ve2 orthologs was used to

identify nucleotides segregating with V. dahliae

resistance or susceptibility using Clustal X (Larkin

et al. 2007). The presence of potential CAPS markers

was tested using the software dCAPS Finder 2.0 (Neff

et al. 2002) and primers designed to amplify the

suitable regions using the software PrimerSelect

(DNAstar). The region surrounding an MfeI restriction

site was amplified using the primers Ve2-184 (50-GGACTCTCAGAGCTTGTTA-30) and Ve2-421rc

(5 0-AAGTTGGAAGAAAGTGAGAGGACC-30).

Amplification conditions were 2 min of initial dena-

turing at 94 �C, followed by 40 cycles of 30 s at 94 �C,

30 s at 57.5 �C, and 45 s at 68 �C, with a final

extension of 5 min at 68 �C. Each reaction consisted

of 19 PCR Master Mix [containing dNTPs (400 nM

each), 19 Taq Polymerase buffer (New England

Biolabs, Ipswich, MA, USA), 0.8 lM each of forward

and reverse primers, and 0.025 units of Taq Polymer-

ase (New England Biolabs]. 5 ll of the reaction was

used as a template in restriction endonuclease diges-

tions with 5 units of MfeI or ClaI (New England

Biolabs) and 19 NEB buffer 4 in a total volume of

20 ll. When ClaI enzyme was used, the reactions

were supplemented with bovine serum albumin

according to the manufacturer’s directions. The reac-

tions were incubated at 37 �C for 5 h and the products

of the digestion resolved by agarose gel electropho-

resis (2 % w/v). Bands were visualized under ultravi-

olet light after ethidium bromide staining of the gel. A

25-bp ladder (Invitrogen) and/or 1-kb ladder (Gener-

ulerTM, Fermentas Corp., Glen Burnie, MD, USA) was

used as size references.

To test the usefulness of the marker, breeding

material from a broad germplasm base that had been

selected for resistance to VW using the seedling dip

assay was used. The material was rated according to

the protocols presented and assigned a phenotype of

Res if the material was healthy or barely diseased (3–4

ratings), Sus if the material was dead or severely

affected (0–2 ratings) or ModRes (those with inter-

mediate values).

In addition, a set of seven commercial lines that are

used by the potato community as controls in screens

for VW resistance were genotyped. This set of

commercial lines has been used in field experiments

during the National Verticillium trials for several

years (use a ref from a NVT). Germplasm was

phenotyped according to protocols in Jansky (2009)

and Bae et al. (2011). In the case of sap values,

resistant clones (Res) were those having less than 200

colony forming units (cfu)/100 ll of sap, susceptible

(Sus) were those having more than 500 cfu/100 ll,

and the remaining clones were called moderately

resistant (ModRes). Symptom data were also used for

phenotyping. Field resistant clones (Res) had less than

20 % of foliage expressing symptoms, susceptible

(Sus) those having more than 50 % of affected foliage;

the remaining clones were rated moderately resistant

(ModRes). To assign genotype ratings, the following

rules were used: RR corresponded to clones in which

DNA was fully digested by the enzyme (homozygous

for the resistance allele); rr corresponded to clones

with DNA that was not digested by the enzyme

(homozygous for the susceptible allele); and Rr

corresponded to heterozygotes. The coefficient of

determination was used to determine the relationship

between this marker and the previous marker of Bae

et al. (2008).

Results

Disease resistance screening of wild Solanum

populations

Verticillium wilt symptoms were apparent in seed-

lings beginning 8 days after inoculation. The results of

the screening of wild Solanum populations with two

different strains of V. dahliae are presented in Table 1.

Accessions within some species that were rated as

resistant according to the data in NRSP6, such as S.

Mol Breeding (2014) 33:465–476 469

123

brevicaule, S. chacoense, and S. microdontum, were

indeed found highly resistant to the pathogen with

normalized disease ratings (i.e. the difference between

the disease ratings of control plants and treated plants)

between 0 and 0.75. Others, such as S. verrucosum (PI

275259), a NRSP6-documented susceptible popula-

tion, and S. candolleanum (ID 210055), previously

called S. bukasovii, a NRSP6-documented resistant

population, were highly susceptible to the pathogen.

Correlation tests between seedling developmental

rating and disease severity indicated that both vari-

ables were not correlated, suggesting that the infection

process was independent of the developmental stage

of the seedlings at the time of inoculation (data not

shown).

The choice of V. dahliae strain appeared to affect

certain species more than others. Examples of this

were S. berthaultii and S. bulbocastanum (PI 255518)

that were rated as resistant when challenged with

strain V18 but were found to be moderately suscep-

tible or susceptible when infected with strain E10

(Table 1). The overall disease severity ratings were

higher in populations inoculated with strain E10

(average disease severity 1.82) than with strain V18

(average disease severity 1.32), although strain V18

was found to cause more symptoms on both of the S.

stoloniferum populations (Table 1). Independent

t tests of the average disease severity ratings of each

isolate in each population indicated that for S.

kurtzianum and S. berthaultii (PI 310981) E10 was

more pathogenic than V18 (p = 0.06 and 0.08

respectively). Conversely, for S. stoloniferum (PI

255533) and S. brevicaule (PI 246536), V18 was

found to cause more symptoms than E10 (p = 0.3 and

p = 0.1 respectively). Analysis of variance of the

entire data set indicated that the isolates did differ in

pathogenicity (F = 0.003). Tukey’s HSD indicated

that the species and populations tested could be

divided into seven groups according to their disease

severity ratings, with the most resistant, S. brevicaule

(PI 246536), and most susceptible, S. candolleanum

(PI 2100550), being statistically different from the rest

(Table 1).

Development of the molecular markers

The disease resistance data set from the root dip assays

was used as the starting point of a broad sequencing

effort to characterize and compare a diagnostic 1.6-kb

region of the Solanum Ve2 gene within each tested

population. This region was previously used to

develop a marker based on S. chacoense-based

resistance (Bae et al. 2008). PCR amplification of

DNA from 10 Solanum species using the primers

C287F1 and C287R1 produced a single fragment of

1,664 bp. Sequencing of this region in all accessions

was performed and aligned against the Ve2 gene from

tomato (Kawchuk et al. 2001) and the potato Ve-like

sequences from resistant C545 and C287 and suscep-

tible V67 (Jansky and Rouse 2000; Bae et al. 2008).

For each accession, sequencing was based on a bulk of

DNA from the six most resistant or six most suscep-

tible plants, based on symptom expression in the

seedling dip assay. Sequence information was

obtained for 14 accessions of the 10 Solanum species

and tested for segregation with resistance or suscep-

tibility to the pathogen. A subset of this sequence

information is presented in Fig. 1. The overall analysis

of the 1,664-bp fragment indicated that at least 37

polymorphic sites (relative to tomato Ve2) were

identified, with 21 of them segregating with resistance

and 16 with susceptibility (data not shown). The

results of the screening indicated that a CAPS marker

could be developed using the restriction enzyme MfeI

at position 351 of the alignment. Primers VE2-184 and

VE2-421rc were designed to amplify this region.

Digestion of VE2-184 and VE2-421rc PCR products

of clones C545, V67, and C287 produced three

fragments of 260 (uncut product), 167, and 93 bp

(digested product) in resistant C545, one fragment

(260-bp uncut product) in susceptible V67, and two

fragments of 167 and 93 bp (digested product) in

resistant C287 (Fig. 2).

A phylogenetic analysis of the sequences showed

that most can be split into two clades, one co-

segregating with resistance (containing species such

as S. brevicaule, S. chacoense, and S. microdontum)

and the other co-segregating with susceptibility

(Fig. 3). A closer look at the sister clades indicated

that resistant populations could be divided further into

three groups according to the allele conformation at

position 351 of the alignment. The first group

comprises the sequences with the MfeI restriction site

and is therefore fully diagnostic for marker-assisted

selection. A second group carries the nucleotide

sequence ATCGAT at a second position in the locus.

The sequence ATCGAT corresponds to the restriction

enzyme ClaI. The third type of polymorphism found

470 Mol Breeding (2014) 33:465–476

123

corresponded to the sequence CAGTTG, which

recognizes a small clade comprised of mostly S.

kurtzianum individuals. Of importance is the presence

of the ClaI site (termed 351ClaI) because this

restriction enzyme can be used to complement the

first marker and therefore encompass a broader range

of species in the selection process. Restriction endo-

nuclease reactions using ClaI confirmed the presence

of the recognition sequences and generated the

expected three bands of 260 (uncut product), 169,

and 91 bp (fully digested product; Table 2). A second

Fig. 1 Multiple sequence alignment of StVe2-like genes from

wild Solanum populations. StVe2 homologs from wild Solanum

sp. were sequenced using primers C287F1 and C287R2 (Bae

et al. 2008); sequence data was aligned using the program

Clustal X (Larkin et al. 2007). Sequences were sorted according

to the relative susceptibility of the Solanum populations to

infection by Verticillium dahliae (Table 1). Polymorphic sites

were detected based on the consensus sequence of the multiple

alignment using the dCAPS Finder software (Neff et al. 2002).

For display purposes the sequence of tomato Ve2 is shown in the

middle of the alignment and it separates resistant sequences

(above) from susceptible ones (below). Regions enclosed by

ovals show the position of the MfeI and ClaI markers

Fig. 2 MfeI cleaved amplified polymorphism. Primers Ve184

and Ve421rc were used to amplify a 237-bp fragment.

Restriction enzyme MfeI was used to selectively cleave the

PCR fragment according to the genotype of the sample. The

figure shows the results of the restriction digestion with

visualization of fragments using ethidium bromide

Mol Breeding (2014) 33:465–476 471

123

ClaI site (termed 380ClaI), co-segregating with

susceptibility, was also found in the sequence analysis

(Fig. 1). Cleavage of this site is expected to produce

three bands (260 bp corresponding to the uncut

product, and two bands, 198 and 62 bp, corresponding

to the fully cleaved product). Restriction endonucle-

ase reactions using ClaI resulted in the expected

inverse pattern of cleavage as was found when the

MfeI enzyme was used (data not shown).

Marker verification

The MfeI marker was tested on a wide array of

germplasm, including commercial cultivars, breeding

lines, and wild potato populations (Table 2). When the

marker was tested on four populations, HC 9 VW4-1

(breeding lines marked 4-1 to 4-26), HC 9 VW4-5

(breeding lines marked 5-1 to 5-14), C545 9 B9

(breeding lines named 7-1 to 7-20), and M9 9 US-W4

Fig. 3 Sequence similarity of Ve2-like homologs. Ve2 homo-

logs from Solanum species were PCR amplified using the

primers C287F1 and C287R1 (Bae et al. 2008). PCR products

were sequenced and individual sequences were aligned to the

canonical known sequence of Solanum lycopersicon VE2, and to

potato VE2 sequences of the breeding lines C287, V67, and

C545. Sequences containing the MfeI marker at position 351 are

shown with a black background and white letters, while

sequences with the alternative ClaI marker at position 351 are

shown with a light gray background and black lettering.

Sequences with the ClaI marker at position 380 are show with a

gray background and white lettering. Sequences that cannot be

determined by any of these markers have a dotted background.

The number to the right of each accession number denotes the

clade of section Petota to which the species belongs

472 Mol Breeding (2014) 33:465–476

123

(breeding lines marked 8-1 to 8-48), which had been

phenotypically pre-selected for resistance against V.

dahliae, the expected marker segregation results were

observed. A detailed summary of the results is as

follows: HC, a haploid (2n = 2x = 24) breeding line

derived from S. chacoense (Jansky 2009), is

Table 2 Germplasm tested

with markers

a Species abbreviations are

ber = S. berthaultii, brc = S.

brevicaule, chc = S.

chacoense, cmm = S.

commersonii, ifd = S.

infundibuliforme, tbr = S.

tuberosum, ver = S.

verrucosum

b RR homozygous resistant,

Rr heterozygous, rr

homozygous susceptible

c Res resistant, Sus

susceptible, ModRes

moderately resistant

Clone/family Germplasma Ploidy Genotype according tob Phenotypec

MfeI 351ClaI

07-29-1 (2 clones) US-W4 9 C545 Diploid RR Rr Res

07-29-5 US-W4 9 C545 Diploid rr rr Sus

07-32-4 US-W4 9 C545 Diploid rr RR Res

08-51-06 US-W4 9 C545 Diploid Rr Rr Res

08-51-12 US-W4 9 C545 Diploid RR RR Res

08-51-13 (2 clones) US-W4 9 C545 Diploid rr rr Res

08-51-15 US-W4 9 C545 Diploid RR RR Res

08-51-16 US-W4 9 C545 Diploid rr RR Sus

08-51-17 US-W4 9 C545 Diploid Rr Rr Sus

A03 US-W4 9 C545 Diploid RR RR Res

A19 US-W4 9 C545 Diploid rr rr Sus

A22 US-W4 9 C545 Diploid Rr RR Res

A27 US-W4 9 C545 Diploid Rr Rr Res

Arma tbr Tetraploid rr rr Res

Atlantic tbr Tetraploid Rr Rr ModRes

C287 tbr, ber Diploid RR Rr Res

C545 tbr, ber, chc Diploid Rr RR Res

C545 9 B9 (20 clones) tbr, chc, ber Diploid RR or Rr RR or Rr Res

Chc 39-7 chc Diploid RR RR Sus

Chc 40-3 chc Diploid Rr Rr Sus

Chc 523-3 chc Diploid RR RR Res

Chc 524-8 chc Diploid RR RR Sus

HC tbr, chc Diploid RR RR ModRes

HC 9 VW4-1 (26 clones) tbr, chc, brc Diploid RR RR Res

HC 9 VW4-5 (14 clones) tbr, chc, brc Diploid RR RR Res

LRC18-21 chc Diploid Rr Rr Res

LRC373-5 tbr, chc Diploid Rr Rr Res

M7 tbr, ifd Tetraploid Rr RR Sus

M9 9 US-W4 (7 clones) ver, cmm Diploid Rr or rr RR, Rr or rr Sus

Ranger Russet tbr Tetraploid RR RR Res

Red Norland tbr Tetraploid rr rr Sus

Reddale tbr Tetraploid Rr Rr Res

Russet Burbank tbr Tetraploid Rr Rr Sus

Russet Norkotah tbr Tetraploid RR RR Sus

Snowden tbr Tetraploid RR RR ModRes

Superior tbr Tetraploid rr rr Sus

US-W4 tbr Diploid rr rr Sus

V67 C545 9 C287 Diploid rr RR Sus

White Pearl tbr, ber Tetraploid Rr RR Sus

XD-3 tbr, chc Diploid Rr Rr Sus

Mol Breeding (2014) 33:465–476 473

123

homozygous for the resistant allele. VW4-1 and VW4-

5, which are clones selected for Verticillium resistance

(breeding lines marked 4-x and 5-x), are both homo-

zygous for the resistant allele. Therefore, all offspring

from these parents are expected to be resistant and

indeed all the breeding lines marked 4-1 to 4-26 and

5-1 to 5-14 were found to be homozygous for the

resistant allele of the marker. These breeding lines

were previously found to be phenotypically resistant to

Verticillium (data not shown). In the case of breeding

lines 7-1 to 7-20, a close 1:1 segregation of the marker

between homozygous resistant and heterozygous sam-

ples was observed (v2 = 0.25). C545 is heterozygous

for the marker while B9 carries the resistant allele of

the marker. These breeding lines had also been selected

for resistance against this pathogen. Lastly, in the cross

between M9 (heterozygous for marker) and US-W4

(susceptible for marker), the samples marked 8-1 to

8-48 were found to carry the susceptible allele of the

marker in most of the samples (Table 2). The marker

was also tested against the progeny of crosses involv-

ing W4 with C545 or C287 and other breeding material

of various origins, finding an overall good correlation

(r2 = 0.52) between the genotyping using this marker

system and that of the previously described Ve marker

(Bae et al. 2008; data not shown).

When the presence of the marker was tested in

tetraploid cultivars, it was found that moderately

resistant clones such as Ranger Russet and Snowden

are homozygous for the resistant allele. Others such as

resistant clone Reddale and moderately resistant

Atlantic were found to carry both of the alleles, while

susceptible cultivars such as Superior and Red Nor-

land were found to be homozygous for the susceptible

allele. On the other hand, opposite results were seen on

moderately susceptible or susceptible cultivars such as

White Pearl and Russet Norkotah, which were found

to carry the resistant allele. Resistant Arma was found

to be homozygous for the susceptible allele (Table 2).

Discussion

Bae et al. (2008) developed a CAPS marker to help in

breeding for resistance to V. dahliae based on the

dominant Ve2 gene. For this purpose, they used the

sequence information from the breeding lines C545,

C287, and V67. These clones were products of

interspecific crosses between S. chacoense and S.

tuberosum. C545 and C287 were selected for resis-

tance to Verticillium wilt, while V67, a hybrid

between C545 and C287, was selected as a susceptible

control (Jansky and Rouse 2000). The Bae et al. (2008)

marker was effective with related germplasm, but did

not correlate well with resistance from other Solanum

species or in tetraploid clones. Consequently, we

aimed to develop a molecular marker with broader

applicability. Previously, Simko et al. (2004) found at

least 11 sequences with homology to Ve1 in Reddale, a

resistant tetraploid commercial clone. The quantita-

tive trait locus tested by Simko et al. (2004) appeared

to explain 65 % of the phenotypic variation, suggest-

ing that a major-effect locus might be linked to the

Solanum tuberosum Ve1 (StVe1) they studied.

The markers we have presented here have broad

applicability. In our experiments we were able to

follow the marker in populations derived from

HC 9 VW4-1/VW4-5 (RR alleles), C545 9 B9

(RR/Rr segregating population), and M9 9 US-W4

(Rr/rr segregating population). HC is a hybrid between

diploid S. tuberosum and S. chacoense (Hamernik

et al. 2009) and VW4-1/VW4-5 are S. brevicaule-

derived clones. As mentioned above, C545, a product

of the Minnesota breeding program, carries S. bertha-

ultii, S. tarijense, S. tuberosum, and S. chacoense

germplasm and resistance to VW appears to be

inherited in a dominant fashion (Jansky et al. 2004;

Lynch et al. 2004). B9 has S. chacoense background

and M9 has S. verrucosum and S. commersonii.

Sequence analysis of Ve2 resistance gene analogs in

Solanum species indicates that restriction endonucle-

ase assays using ClaI and MfeI should recognize the

resistant allelic conformation in many species, with

the exception of S. kurtzianum (Fig. 3). The results of

this study show that species such as S. berthaultii, S.

microdontum, and S. brevicaule can be used as genetic

sources to increase the resistance of commercial

cultivars against V. dahliae.

In order to identify putative species and populations

to examine in our experiments, we looked into

historical information available in the US Potato

Genebank (NRSP6). Accessions were identified

according to the reported resistance or susceptibility

to V. dahliae based on the results of previous

experiments carried out by Anderson in 1983, Corsini

in 1983, Young in 1984, Deahl in 1984, Hanneman in

1986, Mohan in 1990, and Bastia in 2000. From this

information, a subset of wild Solanum species with

474 Mol Breeding (2014) 33:465–476

123

documented resistance or susceptibility was selected

for testing. These experiments were heterogeneous in

nature and a direct comparison of disease ratings was

not available, and different experimental settings

created different standards for comparison of the

accessions. In some cases, the final ratings of one or

more experiments contradicted the results and con-

clusions of other experiments. In our case, care was

given to select those accessions that were consistently

scored as resistant or susceptible across multiple trials.

Nonetheless, our disease screenings results indicated

that some accessions previously rated as immune or

resistant (S. candolleanum PI210055, S. stoloniferum

PI186545, and S. verrucosum PI195170) were exper-

imentally found to be susceptible in our assays

(Table 1). Our results also suggest the need to screen

germplasm with multiple isolates of the pathogen, as

we identified differences in disease ratings when using

different V. dahliae strains. Our data revealed the

expected continuum of responses, varying from very

resistant to very susceptible, and furthermore provide

new germplasm sources in breeding for VW resis-

tance, as species such as S. berthaultii, S. brevicaule,

and S. microdontum were found to express resistance.

All the species tested, with the exception of S.

bulbocastanum and S. tuberosum, belong to clade 4

of section Petota (Spooner et al. 2005; Fajardo and

Spooner 2011) and their nuclei contain only the A

genome. S. bulbocastanum belongs to the clade 1 ? 2

and carries the B genome. The S. tuberosum popula-

tions tested are amphiploid populations (tetraploid in

nature) phylogenetically related to both clades 1 ? 2

and 4 (Spooner et al. 2005, 2010; Fajardo and Spooner

2011). Their nuclei carry both types of genomes (A

and B; Rodriguez et al. 2009). Since Ve was originally

found in tomato and orthologs of this gene are present

in mint and other species, it is tempting to speculate

that resistance to V. dahliae is ancestral to the

evolution of cultivated potato (Fradin et al. 2011).

The fact that resistance is present within clades 1 ? 2

and 4 is an indication that more screening could be

done to pinpoint other species with strong resistance to

this pathogen (likely still dependent on Ve) that

could serve as sources of germplasm for breeding

purposes.

The resistance response observed in some of the

species tested in our experiments was not absolute.

Retrieval of the pathogen from infected tissue was

successful for all the susceptible species shown in

Table 1 and for individuals of S. chacoense and S.

kurtzianum that were rated as resistant (data not

shown). Therefore, tolerance to pathogen infection is a

parameter that should be taken into account when

selecting material for breeding purposes.

Although out of the scope of this publication given

the small number of species tested, we attempted to

correlate resistant populations with a particular geo-

graphical point of origin. This preliminary analysis

indicated that the region comprising the southern edge

of Bolivia and Peru and the northern areas of Chile and

Argentina might contain a hot spot for resistance

against this pathogen, as S. chacoense, S. berthaultii, S.

infundibuliforme, and S. microdontum all originated in

this area (Hijmans and Spooner 2001; Ovchinnikova

et al. 2011). Nonetheless, other species tested, such as

S. boliviense (blv 310974) and S. brevicaule (brv

472974) which were rated moderately susceptible, are

also from these regions in South America. The analysis

also indicated that S. bulbocastanum (blb 275193)

which originated in Mexico also carries resistance (but

not immunity) to this pathogen. Spooner et al. (2009)

carried out tests of taxonomic and biogeographic

predictivity on 10,738 disease and pests evaluations,

encompassing 32 pests and pathogens including V.

dahliae. Their analysis was unable to detect a clear

correlation between geographical point of origin and

resistance to this pathogen (Spooner et al. 2009).

In summary, a molecular marker based on the Ve2

gene was developed. The marker shows promise for

the screening of Verticillium-resistant segregating

populations, in particular those at the diploid level.

Based on the results presented here, wild Solanum

populations might contain alternative sources for

Verticillium resistance linked to a locus that contains

orthologs of Ve.

Acknowledgments Germplasm was provided by the NRSP-6

Potato Genebank. Funding was provided by an ARS-State

Cooperative Potato Research Grant.

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