Advertisement

Molecular Breeding

, Volume 23, Issue 3, pp 377–388 | Cite as

Robust and highly informative microsatellite-based genetic identity kit for potato

  • Marc GhislainEmail author
  • Jorge Núñez
  • María del Rosario Herrera
  • José Pignataro
  • Frank Guzman
  • Merideth Bonierbale
  • David M. Spooner
Open Access
Article

Abstract

The fingerprinting of 742 potato landraces with 51 simple sequence repeat (SSR, or microsatellite) markers resulted in improving a previously constructed potato genetic identity kit. All SSR marker loci were assayed with a collection of highly diverse landraces of all species of cultivated potato with ploidies ranging from diploid to pentaploid. Loci number, amplification reproducibility, and polymorphic information content were recorded. Out of 148 SSR markers of which 30 are new, we identified 58 new SSR marker locations on at least one of three potato genetic linkage maps. These results permitted the selection of a new potato genetic identity kit based on 24 SSR markers with two per chromosome separated by at least 10 cM, single locus, high polymorphic information content, and high quality of amplicons as determined by clarity and reproducibility. The comparison of a similarity matrix of 742 landraces obtained with the 24 SSR markers of the new kit and with the entire dataset of 51 SSR markers showed a high correlation (r = 0.94) by a Mantel test and even higher correlations (r = 0.99) regarding topological comparisons of major branches of a neighbor joining tree. This new potato genetic identity kit is able to discriminate 93.5% of the 742 landraces compared to 98.8% with 51 SSR markers. In addition, we made a marker-specific set of allele size standards that conveniently and unambiguously provide accurate sizing of all alleles of the 24 SSR markers across laboratories and platforms. The new potato genetic identity kit will be of particular utility to standardize the choice and allele sizing of microsatellites in potato and aid in collaborative projects by allowing cumulative analysis of independently generated data.

Keywords

Microsatellite Potato Solanum tuberosum SSR 

Abbreviations

CIP

Centro International de la Papa (International Potato Center)

cM

Centimorgans

EST

Expressed sequence tags

PGI kit

Potato genetic identity kit

PIC

Polymorphic information content

SSR

Simple sequence repeat

Ta

Annealing temperature

TIGR

The Institute of Genomic Research

Introduction

Simple sequence repeats (SSRs, or microsatellites) have been used to great advantage in potato for studies of diversity, genetic structure, and classification (Spooner et al. 2007); tracing germplasm migrations (Spooner et al. 2005a; Rios et al. 2007); fingerprinting (Moisan-Thiery et al. 2005; Provan et al. 1996; Schneider and Douches 1997); genetic linkage mapping (Ghislain et al. 2001; Feingold et al. 2005); establishment of core collections (Ghislain et al. 2006); and investigations of duplicate collections across genebanks (Del Rio et al. 2006). Although not yet used in potato they have potential applications in studies of linkage disequilibrium (Remington et al. 2001; Stich et al. 2005) and gene flow (Devaux et al. 2005; Fenart et al. 2007). They require considerable developmental costs and often have maximum utility within a narrow range of germplasm from which they were developed. Once developed, however, they have tremendous advantages over many other marker classes to include low operational costs, codominance, hypervariablity, high quality bands, highly reproducible bands, amenability to automation, ease of multiplexing, and use with low quality DNA (Spooner et al. 2005b).

Over 200 potato SSRs have been identified through enriched genomic libraries and database searches of expressed sequence tags (ESTs) (Milbourne et al. 1998; Ashkenazi et al. 2001; Ghislain et al. 2004; Feingold et al. 2005). However, many more are becoming available as ESTs are being identified. The latest SSR summary statistics from the former The Institute for Genomic Research (TIGR) document more than 5,800 sequences with potentially useful SSR (repeats of 2–6 nucleotides) markers for potato. These SSRs differ greatly, however, in quality (clarity and repeatability of bands), map location, and polymorphism. Some of them have been tested on potato landraces and advanced varieties mapped on various potato genetic maps. However, an extensive analysis on a large collection of potato SSRs was lacking. Ghislain et al. (2004) provided the first such analysis of 156 SSRs for quality and polymorphism, chose 22 of them by a combination of the above criteria in cultivated potato, showed how some of these could be multiplexed, and mapped them.

The purpose of the present study is to screen additional potato SSRs from all taxonomic groups of potato to refine a selection of microsatellites for maximum utility in a cultivated potato background. Such large data set is available from a previous study aiming at classifying cultivated potato (Spooner et al. 2007). Its wide genetic diversity makes this data set particularly valuable for our purpose.

Materials and methods

Plant materials and DNA extraction

Seven hundred and forty-two native (landrace) potatoes belonging to a composite genotyping set of potato at the International Potato Center (CIP) were used for this study. These landraces were selected to represent all four species and taxonomic groups of potato as described above, and represent the same used in the taxonomic study of Spooner et al. (2007) and are described in the supporting dataset 1. Genomic DNA was obtained using standard protocol derived from Doyle and Doyle (1990). DNA concentration was estimated by using a TBS-380 Fluorometer (Turner BioSystems, USA) with PicoGreen® reagent and 500 ng/ml salmon DNA as reference.

Microsatellite markers and PCR conditions

Eighty-eight SSR markers were obtained from four sources: (1) 22 belong to the previously identified potato genetic identity (PGI) kit (Ghislain et al. 2004), (2) 13 from ESTs developed at the Scottish Crop Research Institute (Milbourne et al. 1998), (3) 30 identified at CIP using the potato EST database of the former The Institute for Genomic Research (http://www.tigr.org/), and (4) 23 from the University of Idaho (Feingold et al. 2005).

PCR reactions were performed in a 10 μl volume containing 100 mM Tris–HCl (Sigma), 20 mM (NH4)2SO4 (Merck), 2.5 mM MgCl2 (Merck), 0.2 mM of each dNTP (Amersham), 25 pM of 700 or 800 IRDye-labeled M13 forward primer (LI-COR), 22 pM M13-tailed forward SSR primer (Invitrogen), 15 pM reverse SSR primer (Invitrogen), 1 unit of Taq polymerase, and 15 ng of genomic DNA. PCR was carried out in a PTC-100 or PTC-200 thermocycler (MJ Research Inc.) using the following cycling profiles: 4 min at 94°C; 33 cycles of 1 min at 94°C, 1 min at annealing temperature (T a) determined experimentally for each SSR primer combination and 1 min at 72°C; with a final extension step of 4 min at 72°C. Blue Stop solution (#830-05630, LI-COR, USA) in a ratio of 1:1 was added to the PCR reaction before loading. PCR products were separated by electrophoresis on a 4300 LI-COR DNA Analyzer system. We sized alleles with the IRDye 50–350 bp fragment size ladder (LI-COR, USA). SSR alleles were detected and scored using the SAGA Generation 2 software (LI-COR, USA).

Mapping new SSR markers

Previously mapped and new SSR markers were mapped on at least one of three segregating diploid populations from which genetic maps were developed: the PD population (Ghislain et al. 2001), BCT population (Bonierbale et al. 1988) or the PCC1 population (Villamón et al. 2005). A total of 148 SSR markers were used in the present map effort and are provided in the supporting dataset 2. The segregation data of 27 SSR marker alleles located on the BCT genetic map was provided to us by the research group (Feingold et al. 2005). Marker alleles segregated as 1:1 ratio whereas skewed markers were rejected using the threshold value established for each genetic map (goodness of fit χ2 test). Null alleles were not considered. Linkage analysis of marker alleles segregating from the respective source parent was performed using JoinMap 3.0 (Stam 1993) with a LOD score of 3.

Polymorphic information content and matrix comparison

SSR marker alleles were scored for presence or absence of the band for all 742 genotypes and treated as dominant marker. The polymorphic information content (PIC) was calculated as PIC = 1−∑(p i 2 ), where p i is the frequency of the ith allele detected in all individuals of the population (Nei 1973). In addition, the ability of a refined set of SSRs chosen here to discriminate a large dataset were compared to a neighbor-joining analysis of Spooner et al. (2007), who analyzed 742 accessions with 50 SSRs by neighbor joining in DARwin software 4.0 (http://darwin.cirad.fr/darwin/Home.php), to which we added one SSR (STM0019). For this analysis, similarity matrixes were calculated using Jaccard’s coefficient and the comparison of the similarity matrixes were performed using the Mantel matrix-correspondence test in the MXCOMP option of the NTSYS 2.02h software (Sokal and Rohlf 1995). Correlations were conducted of the three main branches of this tree (the “bitter potato” (S. ajanhuiri, S. curtilobum, and S. juzepczukii) cluster, the diploid cluster, and the polyploid cluster), using correlation statistics in Microsoft Excel: ρ X,Y  = cov (X,Y)/σXσY, where ρ X,Y is the correlation coefficient (r), cov (X,Y) is the co-variance of X and Y, σ X is the standard deviation of X, and σ y is the standard deviation of Y.

Construction of new potato SSR fragment size ladders

We initially used a pUC18 sequencing reaction or a IRDye-50–350 size standard in our LI-COR DNA Analyzer System as a fragment size ladder. To make our new kit easily applicable to the cultivated potato germplasm base across all platforms, we constructed new size ladders for each of the 24 primer pairs. We examined allele sizes from our database and selected genotypes displaying a range of sizes based on the following three criteria: (1) good separation among the alleles (>3 bp), (2) choice of allele/genotype combinations highlighting the high-frequency alleles encountered in our screening studies, and (3) the presence of the minimum and maximum size of the range of alleles, when possible. Genomic DNA was obtained from the DNA bank of CIP. Amplification products were obtained using standard protocol for SSR markers from CIP (Ghislain et al. 2004). Optimization of PCR conditions were conducted through temperature gradient PCR experiment for optimal annealing temperature, and appropriate number of amplification cycles to obtain good gel resolution of the bands. Electrophoreses to separate amplified products were performed using denaturant 6% poly-acrylamide gels and a silver stain protocol to reveal the bands (Ghislain et al. 2004).

Results

Genetic mapping

The 30 new candidate SSR markers from the TIGR database were surveyed for polymorphism in the PD, BCT and PPC1 mapping populations (details are included in the supporting dataset 2). Two markers were monomorphic in all populations and three markers displayed a skewed segregation from the expected 1:1 ratio. The remaining 25 SSR markers could be mapped in one or two of the three populations using a LOD score of 3. The use of three segregating populations allowed us to identify 33 new map locations of 29 SSR markers not previously mapped. An integrated map was built with the three maps using a map integration function based on mean recombination frequencies and combined LOD scores of the selected sets of loci from each chromosome (Fig. 1). Four markers (STI0012, STM0019, STM0037, STM1053) of the PGI kit were monomorphic in all three segregating population tested and hence were included graphically based on published maps. Only six out of 157 map locations (STG0023, STG0027a, STM0038, STM2022, STM3009, STM51145) produced conflicting map locations on the integrated map and hence were not included. This map represents the most complete SSR potato map developed to date with 138 mapped potato SSR markers at 147 map locations (excluding the four placed graphically and the six conflicting).
Fig. 1

Potato SSR genetic map including the 24 SSR markers of the new PGI kit (bold) on an integrated potato genetic map developed using framework RFLP and SSR genetic maps

Polymorphic information content and matrix comparisons

We analyzed 742 potato landraces of all four cultivated potato species: S. tuberosum Group Andigenum and Group Chilotanum, S. ajanhuiri, S. curtilobum, and S. juzepczukii with 56 SSR markers; 22 from the prior PGI kit (Ghislain et al. 2004) and the 34 most useful of the remaining 66 SSR markers based on marker quality as observed visually on gels. Out of these, five SSR markers appeared to be multi loci based on exceeding allele number considering the ploidy of the plant sample and these were not considered further.

Data obtained with the remaining 51 SSR markers on the 742 potato landraces were analyzed for polymorphic information content (PIC). Considering the cultivated potato a single gene pool (Spooner et al. 2007), markers were scored across all cultivar groups with different ploidies. PIC values per SSR marker ranged from 0.250 to 0.884 while the number of alleles per locus ranged from 2 to 21 (Table 1).
Table 1

Descriptions of the 51 SSR markers and the selected 24 of the new PGI kit by their respective name, source, GenBank accession number, repeat motifs, forward and reverse primer sequences, annealing temperature, map location, allele size and number, and polymorphic information content (PIC) in 742 landraces (Spooner et al. 2007)

Name

Source

Repeat motif

GenBank accession # [SCRI unpublished #]

Primer sequences

T°a

Map location

Size (bp)

# Alleles

PIC

PGI kit

STG0001

g

(CT)n

BE340539

CAgCCAACATTTgTACCCCT ACCCCCACTTgCCATATTTT

58 (52)

XI g

137–163

15

0.699

Yes

STG0004

g

(GT)n

BI434815

TgAAAgCCAATCTCACTggA TATAATTggCTTgCgAgTgC

57 (55)

XI g

212–217

4

0.547

No

STG0006

g

(AC)n

BQ512261

TgAAAACTggTTTCCgCATT TAAgCAAgCTCTCTCCAggg

55 (57)

II g

148–180

9

0.644

No

STG0010

g

(TG)n

BM407152

CgATCTCTgCTTTgCAggTA gTTCATCACTACCgCCgACT

60 (55)

III g

175–192

11

0.685

Yes

STG0014

g

(AAT)n

BQ121319

gAATgTTTATCAgggCAATgg ggAAgATAACAggCACCCAA

54 (53)

VIII g

137–157

7

0.332

No

STG0016

g

(AGA)n

BI178934

AgCTgCTCAgCATCAAgAgA ACCACCTCAggCACTTCATC

55 (53)

I g

137–174

15

0.773

Yes

STG0017

g

(AGT)n

BQ113661

TTAATTggACAgCgTggACA AgCggAACAATgTgTAAggg

55 (55)

144–156

5

0.533

no

STG0021

g

(AAGA)n

BQ506520

TgCCTACTgCCCAAAACATT ACTggCTgggAAgCATACAC

57 (55)

V g

128–166

12

0.79

No

STG0025

g

(AAAC)n

BQ506618

TggAATCCgAATTACgCTCT AggTTTTACCACTCgggCTT

56 (55)

X g

208–223

7

0.531

Yes

STG0026

g

(CTCC)n

BQ119932

ACTgCCgCAAAAAgTgAAAA gCCgCTAggTggAgTAgATg

55 (55)

288–308

7

0.48

No

STG0032

g

(GCA)n

BQ121606

TTCATTTTCCgTTCAAAgCC TCgAgATCCATCATCAACgA

57 (53)

VIII g

127–136

6

0.373

No

STG0033

g

(CGG)n

BI935647

gCTCATTTgACTgCTAAACCC gAAAgAATTgTgCCgTCgAT

56 (55)

II g

148–162

6

0.661

No

STI0001

f

(AAT)n

CK860917

CAgCAAAATCAgAACCCgAT ggATCATCAAATTCACCgCT

60 (55)

IV fg

194–215

8

0.688

Yes

STI0003

f

(ACC)n

AW096896

ACCATCCACCATgTCAATgC CTCATggATggTgTCATTgg

60 (55)

VIII fg

137–188

17

0.746

Yes

STI0004

f

(AAG)n

BQ118939

GCTgCTAAACACTCAAgCAgAA CAACTACAAgATTCCATCCACAg

60 (55)

VI fg

83–126

13

0.688

Yes

STI0012

f

(ATT)n

U69633

gAAgCgACTTCCAAAATCAgA AAAgggAggAATAgAAACCAAAA

56 (55)

IV f

183–234

15

0.791

Yes

STI0014

f

(TGG)n (AGG)n

BQ115461

AgAAACTgAgTTgTgTTTgggA TCAACAgTCTCAgAAAACCCTCT

54 (55)

IX fg

127–157

10

0.708

Yes

STI0019

f

(ATCT)n

EG013494

TCCCTgTTgCCTTgAACAAT TgggAAAAggTACAAAgACgA

61 (60)

VII f

135–149

11

0.709

No

STI0022

f

(ACCCG)n

BQ511964

TCTCCAATTACTTgATggACCC CAATgCCATACACgTggCTA

61 (60)

VIII fg

126–156

10

0.713

No

STI0023

f

(CAG)n

BI920780

gCgAATgACAggACAAgAgg TgCCACTgCTACCATAACCA

61 (60)

X fg

172–245

21

0.813

No

STI0025

f

(CTCC)n

BQ119932

CTgCCgCAAAAAgTgAAAAC TgAATgTAggCCAAATTTTgAA

56 (60)

VII f

115–135

6

0.454

No

STI0030

f

(ATT)n

BF188393

TTgACCCTCCAACTATAgATTCTTC TgACAACTTTAAAgCATATgTCAgC

58 (60)

XII fg

94–137

17

0.811

Yes

STI0032

f

(GGA)n

BQ120452

TgggAAgAATCCTgAAATgg TgCTCTACCAATTAACggCA

61 (60)

V fg

127–148

8

0.745

Yes

STI0033

f

(AGG)n

BG886969

TgAgggTTTTCAgAAAgggA CATCCTTgCAACAACCTCCT

61 (60)

VII fg

131–155

9

0.512

Yes

STI0035

f

(TTC)n

BE919921

ACCTTTgAggAATTgCAggA CATTgAAggAgTTCCAgTCC

60 (57)

108–124

6

0.568

No

STI0036

f

(AC)n (TC)imp

BI920053

ggACTggCTgACCATgAACT TTACAggAAATgCAAACTTCg

55 (55)

II g

129–164

17

0.806

No

STI0038

f

(CTG)n

BQ509486

CCAAATgAggCTAAgggTgA ggCCAAgAAAATCAAgAACg

60 (58)

V g

112–127

7

0.546

No

STM0019a,b

d

(AT)n (GT)n (AT)n (GT)n (GC)n (GT)n

[PAC33]

AATAggTgTACTgACTCTCAATg TTgAAgTAAAAgTCCTAgTATgTg

– (47)

VI dg

(99–206)

17, 13

0.826

Yes

STM0030

d

compound(GT/GC) (GT)n

[PAC05]

AgAgATCgATgTAAAACACgT gTggCATTTTgATggATT

58 (53)

XII deg

122–168

19

0.868

No

STM0031

d

(AC)n…(AC)n GCAC (AC)n (GCAC)n

[PAC50]

CATACgCACgCACgTACAC TTCAACCTATCATTTTgTgAgTCg

53 (57)

VII dg

185–211

10

0.721

Yes

STM0037

d

(TC)n (AC)n AA (AC)n (AT)n

[PAC62]

AATTTAACTTAgAAgATTAgTCTC ATTTggTTgggTATgATA

52 (53)

XI dg

87–133

17

0.778

Yes

STM1016

d

(TCT)n

EU548082

TTCTgATTTCATgCATgTTTCC ATgCTTgCCATgTgATgTgT

56 (56)

VIII eg

243–275

17

0.84

No

STM1017

d

(ATT)n

L13771

gACACgTTCACCATAAAA AgAAgAATAgCAAAgCAA

– (53)

IX d

(132–136)

2

0.25

No

STM1031

d

(AT)n

X55748

TgTgTTTgTTTTTCTgTAT AATTCTATCCTCATCTCTA

– (55)

V dg

(236–301)

19

0.499

No

STM1049

d

(ATA)n

X13497

CTACCAgTTTgTTgATTgTggTg AgggACTTTAATTTgTTggACg

54 (57)

I dg

197–219

9

0.543

No

STM1052

d

(AT)n GT (AT)n (GT)n

AJ133765

CAATTTCgTTTTTTCATgTgACAC ATggCgTAATTTgATTTAATACgTAA

50 (52)

IX eg

214–263

17

0.832

Yes

STM1053

d

(TA)n (ATC)n

AB022690

TCTCCCCATCTTAATgTTTC CAACACAgCATACAgATCATC

53 (53)

III dg

170–196

10

0.688

Yes

STM1058

d

(ATT)n

Z13992

ACAATTTAATTCAAgAAgCTAgg CCAAATTTgTATACTTCAATATgA

55 (55)

III dg

130–139

4

0.285

No

STM1064

d

(TA)n (TG)n GT (TG)n

AC215425

gTTCTTTTggTggTTTTCCT TTATTTCTCTgTTgTTgCTg

55 (55)

II deg

201–213

8

0.566

Yes

STM1104

d

(TCT)n

EU548082

TgATTCTCTTgCCTACTgTAATCg CAAAgTggTgTgAAgCTgTgA

53 (57)

VIII deg

178–199

14

0.884

Yes

STM1106

d

(ATT)n

X95821

TCCAgCTgATTggTTAggTTg ATgCgAATCTACTCgTCATgg

51 (55)

X dg

145–211

17

0.821

Yes

STM2022

d

(CAA)n…(CAA)n

[C112]

gCgTCAgCgATTTCAgTACTA TTCAgTCAACTCCTgTTgCg

58 (53)

II deg

173–243

7

0.621

No

STM2030

d

(CA)n (TA)n

[C216]

TCTTCCCAAATCTAgAATACATgC AAAgTTAgCATggACAgCATTTC

– (55)

I d

(180–209)

4

0.274

No

STM3012

d

(CT)n (CT)n

[61D9]

CAACTCAAACCAgAAggCAAA gAgAAATgggCACAAAAAACA

56 (57)

IX d

180–225

9

0.65

No

STM5114

h

(ACC)n

[p102B19]

AATggCTCTCTCTgTATgCT gCTgTCCCAACTATCTTTgA

60 (57)

II eg

297–322

11

0.693

Yes

STM5121

h

(TGT)n

[p46L17]

CACCggAATAAgCggATCT TCTTCCCTTCCATTTgTCA

48 (50)

XII g

297–309

8

0.733

Yes

STM5127

h

(TCT)n

[p23e7]

TTCAAgAATAggCAAAACCA CTTTTTCTgACTgAgTTgCCTC

55 (60)

I eg

248–291

17

0.853

Yes

STM5140

h

(AAT)n

[p10B1]

gCTATTgTTgCAgATAATACg gCCATgCACTAATCTTTgATTC

60 (57)

IV e

180–219

12

0.674

No

STPoAc58

e

(TA)n

X55749

TTgATgAAAggAATgCAgCTTgTg ACgTTAAAgAAgTgAgAgTACgAC

– (57)

V eg

(243–263)

13

0.754

Yes

STGBSS

c

(TCT)n

EU548082

AATCggTgATAAATgTgAATgC ATgCTTgCCATgTgATgTgT

53 (53)

VIII eg

121–150

16

0.844

No

STWAX-2

a

(ACTC)n

EU548082

CCCATAATACTgTCgATgAgCA gAATgTAgggAAACATgCATgA

53 (53)

VIII eg

228–254

15

0.784

No

Data are provided for bands produced with M13-tailed PCR primers using the 4300 LI-COR DNA Analyzer System except those with parenthesis which follow Ghislain et al. (2004). Only one marker, STM0019 amplifies two loci. The source and map location of the SSR markers are indicated with a, for Veilleux et al. (1995); b, for Kawchuk et al. (1996); c, for Provan et al. (1996); d, for Milbourne et al. (1998); e, for Ghislain et al. (2004); f, for Feingold et al. (2005); g, for this article; and h, for unpublished from SCRI. Last column indicates whether the SSR marker pertains to the new PGI kit

The discriminatory capacity of the SSR markers was analyzed by comparing a similarity matrix generated on 742 genotypes analyzed with 8, 16, 24, 32, 42 and 51 SSR markers ranked by their PIC value (Fig. 2). The results indicated that the 24 SSR markers with the highest PIC values provided nearly identical similarity matrices with one generated with the 32 highest PIC values (r = 0.97). In total, 93.5% of the genotypes can be discriminated using the selected 24 SSR markers (Table 1). However, comparison of placement of accessions within the three main branches of the 742 accession neighbor-joining tree of Spooner et al. (2007) were correlated at r = 0.99, suggesting that the 6.5% of the accessions not absolutely discriminated using the 24 markers are all so similar as to have little effect on major groups discovered in phenetic or phylogenetic analyses. These results lead us to propose 24 (Table 1) as an appropriate number of SSR markers for a new PGI kit.
Fig. 2

Validation of the 24 SSR markers selected by discrimination analysis using genotyping data of 742 landraces: a Comparison of similarity matrixes generated with 8, 16, 24, 32, 42 and 51 SSR markers by r-values (▲), and by percentage of discrimination (■). b Representation of the comparison of similarity matrixes generated by 24 SSR [Y label] markers of the new PGI kit and the 51 SSR markers [X label] and the corresponding correlation coefficient r

Selection of the new PGI kit

The most informative 24 SSR markers for genotyping potato landraces were selected based on quality criteria, genome coverage, and locus-specific information content. We selected two SSR markers per chromosome with a linkage distance at least 10 cM except for chromosome VII, where markers STM0031 and STI0033 were separated by only 3 cM due to lack of alternative markers with a high PIC value (Fig. 1).

New potato SSR fragment size ladders

To construct a fragment size ladder for each SSR marker, we chose the alleles that were: (1) high-frequency, (2) covered the range of allele sizes, (3) well-separated alleles while avoiding those giving overlapping bands due to stuttering, and (4) displayed by a minimum number of landraces. These provide effective size ladders for easy extrapolation of alleles not part of the size kit. Annealing temperatures had to be verified and for few cases adjusted. Final DNA concentrations of the selected genotype range between 3 and 12 ng/μl to be mixed to produce the ladder (Table 2). We succeed in identifying a maximum of four (e.g., STM0019) genotypes to have good coverage of allele sizes. The number of alleles for each of the 24 SSR fragment size standard ranged from three (STM5121) to nine (STI0012) with an average number of 5.5 (Table 2). Overall, the 24 SSR fragment size standards produce 137 reference alleles, representing 44.7% of the total of 306 alleles found in 742 accessions by the 24 SSR markers of the new PGI kit. Allele sizes included in the size standards ranged from 83 to 322 bp providing an easy and convenient tool for identification of allele sizes (Fig. 3).
Table 2

SSR fragment size standard for each SSR marker of the PGI kit

Locus

T°a

Accession

DNA (ng/μl)

Alleles

Size standards alleles

STG0001

58

705710

706722

705029

3

154 150 146 142

136

163 146

136 142 146 150 154 163

STG0010

58

703494

705056

6

3

192 187

192 182 177

177 182 187 192

STG0016

55

705191

704414

702870

9

160 154 148 143

154 137

172 143

137 143 148 154 160 172

STG0025

56

704434

705903

3

219 215 208

223 215

208 215 219 223

STl0001

60

704148

704236

3

212 206 194

200

194 200 206 212

STI0003

60

703721

703284

704149

9

158 137

170 158 149

188 179 170 158

137 149 158 170 179 188

STI0004

60

704019

703882

702646

704309

3

101 95 92

121 107 95

95 92 83

112 95

83 92 95 101 107 112 121

STI0012

56

704469

705234

703381

706032

12

234 209 204 191

201 189 186 183

207 204 201 183

189 234

183 186 189 191 201 204 207 209 234

STI0014

54

704669

705029

3

154 148 145 139

148 145 133

133 139 145 148 154

STI0030

58

705772

705054

705958

3

137 118 109

104 125

132 109

104 109 118 125 132 137

STI0032

61

705191

705739

3

130 136

127 136 142 148

127 130 136 142 148

STI0033

61

705111

706134

3

155 149 137 131

143

131 137 143 149 155

STM0019a

(47)

703783

704717

705116

704074

12

(168 213)

(161 194)

(201 206)

(176 184 213)

(161 168 176 184 194 201 206 213)

STM0019b

(47)

701399

702937

705211

9

(93 99 119)

(93 97 103)

(93 107)

(93 97 103 107 119)

STM0031

56

704592

705875

6

203 195 185

211

185 195 203 211

STM0037

48

705211

703296

704073

9

87 91

91 101 107

91 133

87 91 101 107 133

STM1052

50

703558

705874

705601

704472

12

226 235 262

214 226 235 243

226 256

235 243 250 256

214 226 235 243 250 256 262

STM1053

52

705887

703721

705811

3

192 189 180

189 186 170

196 186

170 180 186 189 192 196

STM1064

52

706702

700299

6

209 206 201

213 209 206

201 206 209 213

STM1104

55

704835

703493

7.5

186 190 195

178 190 199

178 186 190 195 199

STM1106

49

704368

703502

705964

9

157 163

169 175 181 211

145

145 157 163 169 175 181 211

STM5114

60

704903

704183

3

314 308 302

322 314

302 308 314 322

STM5121

50

706643

703759

 

297 309

303

297 303 309

STM5127

52

706776

705655

3

285 276 269 263 248

291 269 266 258

248 258 263 266 269 276 285 291

STPoAc58

(57)

704166

702824

6

263 249

253 249 243

243 249 253 263

Data are provided for bands produced with M13-tailed PCR primers using the 4300 LI-COR DNA Analyzer System except those with parenthesis which follow Ghislain et al. (2004). Potato accessions are presented by their CIP genebank numbers recommended template DNA concentration and the alleles produced

Fig. 3

SSR fragment size standard for the SSR marker STM5127 displaying eight well-defined and spaced alleles using a mix of only 2-genotypes

Discussion

The new PGI kit is composed of 24 SSR markers from over 200 we screened. It provides high-quality, high polymorphism alleles with two markers from each of the 12 linkage groups of potato separated by at least 10 cM, except for chromosome VII with two SSRs separated by only 3 cM. It discriminates representative germplasm samples from all potato cultivar groups with high accuracy. Nine SSRs are from the previous PGI kit (Ghislain et al. 2004), three from ESTs developed at SCRI, four from TIGR, and eight from the University of Idaho. A composite reference DNA sample can conveniently be used to provide accurate sizing of all alleles for these SSR markers across laboratories and platforms.

The PGI kit can be used for potato germplasm characterization for a variety of purposes from identity verification (fingerprinting), to studies of genetic diversity, anchoring genetic linkage maps, establishment of core collections, and gene flow. The 24 composite reference samples of DNA for allelic size determinations will stimulate and foster collaborations worldwide on the use of SSRs for these applications. For example, we used the new PGI kit to identify potential duplicate landraces between the CIP and PROINPA Bolivian potato germplasm collections (data not shown). Some landraces belong to the same morphologically selected cluster were not grouped into the same molecular cluster especially for landraces of very diverse germplasm sets such as the S. tuberosum Andigenum Group. There was a total correspondence with the less diverse S. tuberosum Chilotanum Group. In another application, the new PGI kit has been using to genotype breeding lines and advanced cultivars of potato, and most of the breeding material groups into a well-defined cluster with landraces of the Chilotanum group (data not shown). Such grouping is expected because the germplasm of the Chilotanum group has been used extensively in potato breeding worldwide.

In summary, these highly characterized new SSR markers have tremendous utility for a variety of applications and can stimulate standardization and international collaborations within the cultivated potato gene pool. The PGI kit including primers and fragment size standard are available upon request. A SSR database of the cultivated potato is available on line from the bioinformatics portal of the Generation Challenge Program web site (www.generationcp.org) and of CIP (http://research.cip.cgiar.org/confluence/display/IPD/SSR+Marker). The latter provides a full description of each SSR markers, amplification and detection conditions, and the genotyping data of all potato landraces available to date. It is expected that with increased uses the SSR database will be integrated with the germplasm database of the CGIAR centers.

Notes

Acknowledgments

We thank Sergio Feingold and Jim Lorenzen for providing their data set of the map location of SSR markers. This research was supported by the Grant SP1C2-2004-5 from the Generation Challenge Program, the International Potato Center, and by U.S. National Science Foundation Division of Environmental Biology (NSF DEB) 0316614 entitled: A world-wide treatment of Solanum (http://www.nhm.ac.uk/research-curation/projects/solanaceaesource//). The authors are grateful to the conscious dedication of Luciano Fernandez for his technical support.

Open Access

This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited.

Supplementary material

11032_2008_9240_MOESM1_ESM.xls (5.8 mb)
(XLS 5903 kb)
11032_2008_9240_MOESM2_ESM.xls (50 kb)
(XLS 50 kb)

References

  1. Ashkenazi V, Chani E, Lavi U, Levy D, Hillel J, Veilleux RE (2001) Development of microsatellites markers in potato and their use in phylogenetic and fingerprinting analysis. Genome 44:50–62. doi: 10.1139/gen-44-1-50 PubMedCrossRefGoogle Scholar
  2. Bonierbale MW, Plaisted RL, Tanksley SD (1988) RFLP maps based on a common set of clones reveal modes of chromosomal evolution in potato and tomato. Genetics 120:1095–1103PubMedGoogle Scholar
  3. Del Rio A, Bamberg J, Huamán Z (2006) Genetic equivalence of putative duplicate germplasm collections held at CIP and US potato genebanks. Am J Potato Res 83:279–285CrossRefGoogle Scholar
  4. Devaux C, Lavigne C, Falentin-Guyomarc’h H, Vautrin S, Lecomte JE, Klein K (2005) High diversity of oilseed rape pollen clouds over an agro-ecosystem indicates long-distance dispersal. Mol Ecol 14:2269–2280. doi: 10.1111/j.1365-294X.2005.02554.x PubMedCrossRefGoogle Scholar
  5. Doyle JJ, Doyle JL (1990) Isolation of plant DNA from fresh tissue. BRL Focus 12:13–15Google Scholar
  6. Feingold S, Lloyd J, Norero N, Bonierbale M, Lorenzen J (2005) Mapping and characterization of new EST-derived microsatellites for potato (Solanum tuberosum L.). Theor Appl Genet 111:456–466. doi: 10.1007/s00122-005-2028-2 PubMedCrossRefGoogle Scholar
  7. Fenart S, Austerlitz F, Cuguen J, Arnaud J-F (2007) Long distance pollen-mediated gene flow at a landscape level: the weed beet as a case study. Mol Ecol 16:3801–3813. doi: 10.1111/j.1365-294X.2007.03448.x PubMedCrossRefGoogle Scholar
  8. Ghislain M, Trognitz B, del Rosario Herrera M, Solis J, Casallo G, Vásquez C et al (2001) Genetic loci associated with field resistance to late blight in offspring of Solanum phureja and S. tuberosum grown under short-day conditions. Theor Appl Genet 103:433–442. doi: 10.1007/s00122-001-0545-1 CrossRefGoogle Scholar
  9. Ghislain M, Spooner DM, Rodríguez F, Villamón F, Núñez J, Vásquez C et al (2004) Selection of highly informative and user-friendly microsatellites (SSRs) for genotyping of cultivated potato. Theor Appl Genet 108:881–890. doi: 10.1007/s00122-003-1494-7 PubMedCrossRefGoogle Scholar
  10. Ghislain M, Andrade D, Rodríguez F, Hijmans RJ, Spooner DM (2006) Genetic analysis of the cultivated potato Solanum tuberosum L. Phureja Group using RAPDs and nuclear SSRs. Theor Appl Genet 113:1515–1527. doi: 10.1007/s00122-006-0399-7 PubMedCrossRefGoogle Scholar
  11. Kawchuk LM, Lynch DR, Thomas J, Penner B, Sillito D, Kulcsar F (1996) Characterization of Solanum tuberosum simple sequence repeats and application to potato cultivar identification. Am Potato J 73:325–335. doi: 10.1007/BF02849164 CrossRefGoogle Scholar
  12. Milbourne D, Meyer R, Collins A, Ramsay L, Gebhardt C, Waugh R (1998) Isolation, characterization and mapping of simple sequence repeat loci in potato. Mol Gen Genet 259:233–246. doi: 10.1007/s004380050809 PubMedCrossRefGoogle Scholar
  13. Moisan-Thiery M, Marhadour S, Kerlan MC, Dessenne N, Perramant M, Gokelaere T et al (2005) Potato cultivar identification using simple sequence repeats markers (SSR). Potato Res 48:191–200. doi: 10.1007/BF02742376 CrossRefGoogle Scholar
  14. Nei M (1973) Analysis of gene diversity in subdivided populations. Proc Natl Acad Sci USA 70:3321–3323. doi: 10.1073/pnas.70.12.3321 PubMedCrossRefGoogle Scholar
  15. Provan J, Powell W, Waugh R (1996) Microsatellite analysis of relationships within cultivated potato (Solanum tuberosum). Theor Appl Genet 92:1078–1084. doi: 10.1007/BF00224052 CrossRefGoogle Scholar
  16. Remington DL, Thornsberry JM, Matsuoka Y, Wilson LM, Whitt SR, Doebley J et al (2001) Structure of linkage disequilibrium and phenotypic associations in the maize genome. Proc Natl Acad Sci USA 98:11479–11484. doi: 10.1073/pnas.201394398 PubMedCrossRefGoogle Scholar
  17. Rios D, Ghislain M, Rodriguez F, Spooner DM (2007) What is the origin of the European potato? Evidence from Canary Island landraces. Crop Sci 47:1271–1280CrossRefGoogle Scholar
  18. Schneider K, Douches DS (1997) Assessment of PCR-based simple sequence repeats to fingerprint North American potato cultivars. Am Potato J 74:149–160. doi: 10.1007/BF02851594 CrossRefGoogle Scholar
  19. Sokal RR, Rohlf FJ (1995) Biometry, 3rd edn. Freeman, New York, 0 7167 2411 1Google Scholar
  20. Spooner DM, Núñez J, Rodríguez F, Naik PS, Ghislain M (2005a) Nuclear and chloroplast DNA reassessment of the origin of Indian potato varieties and its implications for the origin of the early European potato. Theor Appl Genet 110:1020–1026. doi: 10.1007/s00122-004-1917-0 PubMedCrossRefGoogle Scholar
  21. Spooner DM, van Treuren RR, de Vicente MC (2005b) Molecular markers for germplasm and genebank management. Tech Bull 10. International Plant Genetic Resources Institute, RomeGoogle Scholar
  22. Spooner DM, Núñez J, Trujillo G, del Rosario Herrera M, Guzmán F, Ghislain M (2007) Extensive simple sequence repeat genotyping of potato landraces supports a major reevaluation of their gene pool structure and classification. Proc Natl Acad Sci USA 104:19398–19403. doi: 10.1073/pnas.0709796104 PubMedCrossRefGoogle Scholar
  23. Stam P (1993) Construction of integrated genetic linkage maps by means of a new computer package: JoinMap. Plant J 3:739–744CrossRefGoogle Scholar
  24. Stich B, Melchinger AE, Frisch M, Maurer HP, Heckenberger M, Reif JC (2005) Linkage disequilibrium in European elite maize germplasm investigated with SSRs. Theor Appl Genet 111:723–730. doi: 10.1007/s00122-005-2057-x PubMedCrossRefGoogle Scholar
  25. Veilleux RE, Shen LY, Paz MM (1995) Analysis of the genetic composition of anther-derived potato by randomly amplified polymorphic DNA and simple sequence repeats. Genome 38:1153–1162PubMedGoogle Scholar
  26. Villamón FG, Spooner DM, Orillo E, Mihovilovich E, Pérez W, Bonierbale MW (2005) Late blight resistance linkages in a novel cross of the wild potato species Solanum paucissectum (series Piurana). Theor Appl Genet 111:1201–1214. doi: 10.1007/s00122-005-0053-9 PubMedCrossRefGoogle Scholar

Copyright information

© The Author(s) 2008

Open AccessThis is an open access article distributed under the terms of the Creative Commons Attribution Noncommercial License (https://creativecommons.org/licenses/by-nc/2.0), which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited.

Authors and Affiliations

  • Marc Ghislain
    • 1
    Email author
  • Jorge Núñez
    • 1
  • María del Rosario Herrera
    • 1
  • José Pignataro
    • 1
  • Frank Guzman
    • 1
  • Merideth Bonierbale
    • 1
  • David M. Spooner
    • 2
  1. 1.Applied Biotechnology Laboratory, International Potato Center, (CIP)Lima 12Peru
  2. 2.USDA-ARS, Vegetable Crops Research Unit, Department of HorticultureUniversity of WisconsinMadisonUSA

Personalised recommendations