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Extensive simple sequence repeat genotyping of
potato landraces supports a major reevaluation of
their gene pool structure and classification
David M. Spooner*†, Jorge Nunez‡, Guillermo Trujillo‡, Marıa del Rosario Herrera‡, Frank Guzman‡, and Marc Ghislain‡
                           ´˜                             ´                                  ´
*Vegetable Crops Research Unit, Agricultural Research Service, U.S. Department of Agriculture, Department of Horticulture, University of Wisconsin, 1575
Linden Drive, Madison, WI 53706-1590; and ‡Applied Biotechnology Laboratory, International Potato Center, Apartado 1558, Lima 12, Peru

Communicated by S. J. Peloquin, University of Wisconsin, Madison, WI, October 16, 2007 (received for review August 15, 2007)

Contrasting taxonomic treatments of potato landraces have con-                   grown nearly as extensively as S. tuberosum, as outlined by
tinued over the last century, with the recognition of anywhere                   Huaman and Spooner (1).
                                                                                        ´
from 1 to 21 distinct Linnean species, or of Cultivar Groups within                 The relationships and extent of genetic differentiation be-
the single species Solanum tuberosum. We provide one of the                      tween the Andean and Chilean landraces has long been contro-
largest molecular marker studies of any crop landraces to date, to               versial. Based on cytoplasmic sterility factors, geographical
include an extensive study of 742 landraces of all cultivated species            isolation, and ecological differences, Grun (9) suggested that
(or Cultivar Groups) and 8 closely related wild species progenitors,             Chilean landraces were distinct from Andean landraces. Hawkes
with 50 nuclear simple sequence repeat (SSR) (also known as                      (2) distinguished the tetraploid Chilean from Andean landraces
microsatellite) primer pairs and a plastid DNA deletion marker that              by characters of the leaf and flower pedicel. Plastid restriction
distinguishes most lowland Chilean from upland Andean land-                      site data documented five genotypes (A, C, S, T, and W types)
races. Neighbor-joining results highlight a tendency to separate                 in the diploid and tetraploid Andean landraces, and the Chilean
three groups: (i) putative diploids, (ii) putative tetraploids, and (iii)        landraces had three types, A, T, and W (10, 11). The most
the hybrid cultivated species S. ajanhuiri (diploid), S. juzepczukii             frequently observed type in the Chilean landraces (21 of 24 or
(triploid), and S. curtilobum (pentaploid). However, there are many              87.5% of the accessions examined) is type T, which is charac-
exceptions to grouping by ploidy. Strong statistical support occurs              terized by a 241-bp deletion (12). Conversely, 5 of the 113 (4.4%)
only for S. ajanhuiri, S. juzepczukii, and S. curtilobum. In combina-            accessions of S. tuberosum subsp. andigenum had the T type
tion with recent morphological analyses and an examination of the                (10–12).
identification history of these collections, we support the reclas-                  Potato landraces have been classified into 21 species (13, 14),
sification of the cultivated potatoes into four species: (i) S. tubero-           7 species with seven subspecies (2) and 9 species with two
sum, with two Cultivar Groups (Andigenum Group of upland                         subspecies (15, 16), or as the single species S. tuberosum with 8
Andean genotypes containing diploids, triploids, and tetraploids,                user-defined Cultivar Groups (1). Cultivar Groups are taxo-
and the Chilotanum Group of lowland tetraploid Chilean land-                     nomic categories used by the International Code of Nomencla-
races); (ii) S. ajanhuiri (diploid); (iii) S. juzepczukii (triploid); and (iv)   ture of Cultivated Plants to associate cultivated plants with traits
S. curtilobum (pentaploid). For other classifications, consistent and             that are of use to agriculturists and are not meant to represent
stable identifications are impossible, and their classification as                 natural groups or species in any classification philosophy. Ploidy
species is artificial and only maintains the confusion of users of the            levels in cultivated potatoes range from diploid (2n 2x 24),
gene banks and literature.                                                       to triploid (2n     3x     36), to tetraploid (2n     4x     48), to
                                                                                 pentaploid (2n 5x 60). Huaman and Spooner (1) examined
                                                                                                                       ´
cultivated   microsatellites   sect. Petota   Solanum tuberosum      taxonomy
                                                                                 the morphological support for the various classifications of
                                                                                 potato landraces using representatives of all seven species from

T   he cultivated potato represents one of the most important
    food plants worldwide, yet interpretation of its gene pool
structure remains controversial. Contrasting taxonomic treat-
                                                                                 the classification of Hawkes (2). The results showed some
                                                                                 morphological support for S. ajanhuiri, S. chaucha, S. curtilobum,
                                                                                 and S. juzepczukii, lesser support for S. tuberosum subsp. tubero-
ments of the landraces have continued over last century, with the                sum, and no support for S. phureja and S. stenotomum. Whatever
recognition of anywhere from 1 to 21 distinct Linnean species,                   morphological support for these entities was present was only by
or of various Cultivar Groups within the single species S.                       using a suite of characters, all of which are shared with other taxa
tuberosum (1). For consistency in usage in our article and to                    (polythetic support). These results, combined with their likely
maintain the names most familiar to scientists, we use the seven                 hybrid origins, multiple origins, and evolutionary dynamics of
species terminology of Hawkes (2). Indigenous cultivated (land-                  continuing hybridization, led Huaman and Spooner (1) to rec-
                                                                                                                         ´
race) potatoes are widely distributed in the Andes from western                  ognize all landrace populations of cultivated potatoes as a single
Venezuela, south to northern Argentina, and with another set of                  species, S. tuberosum, with the eight Cultivar Groups: Ajanhuiri
landraces in south-central Chile in Chiloe Island and the adja-
                                            ´                                    Group, Andigenum Group, Chaucha Group, Chilotanum
cent Chonos Archipelago. The Chilean landraces, although once                    Group, Curtilobum Group, Juzepczukii Group, Phureja Group,
proposed to have arisen independently from central Chile (3),                    and Stenotomum Group (the latter containing all landraces of
are secondarily derived from the Andean ones (2), likely after                   the Goniocalyx Group).
hybridization with the Bolivian and Argentinean species Sola-
num berthaultii (4), a species recently combined with the for-
merly recognized wild species S. tarijense (5). Three of the                     Author contributions: J.N., G.T., M.d.R.H., F.G., and M.G. performed research; and D.M.S.
Andean-cultivated species are hypothesized to be of hybrid                       wrote the paper.
origins with cultivated potatoes and wild species: S. ajanhuiri [S.              The authors declare no conflict of interest.
stenotomum cultivated        S. megistacrolobum wild (6)], S. juz-               †To   whom correspondence should be addressed. E-mail: david.spooner@ars.usda.gov.
epczukii [S. stenotomum        S. acaule wild (7, 8)], and S. curti-             This article contains supporting information online at www.pnas.org/cgi/content/full/
lobum [S. andigenum cultivated S. juzepczukii (7, 8)]. The latter                0709796104/DC1.
three ‘‘bitter potatoes’’ are grown in upland habitats and are not               © 2007 by The National Academy of Sciences of the USA



19398 –19403     PNAS      December 4, 2007      vol. 104   no. 49                                                 www.pnas.org cgi doi 10.1073 pnas.0709796104
48
                                                                                                                                                                     X
                                                                                                                                                   T                                                      48
                                                                                                                                                                                            48

                                                                                                                                                                                       48    48
                                                                                                                                                                                                                    48
                 A                                                                                T
                                                                                                                     T
                                                                                                                                   48
                                                                                                                                                                                                                   phu
                                                                                                                                                       36

                                                                                                        T                                    cur        36
                                                                                                         T
                                                                                                                 T
                                                                                                                     T

                                                                                                      T
                                                                                                             48
                                                                                                                     48


                                                                                                      X
                                                     T            48
                 X                                                                 T
                                                                                          48
                                                                            T                 T


                                                                      X                                                                                                                63
                      All T except                                                                                                                                       52
                      2 as X       X                                                                                                                                           75
                                                                                                                                                                89
                                                                                                                                                                      100

                                                                  X



                                                             T



                                                     p
                                           g   rou
                                       ule                                              jh)
                              ic   a                                           i   (a
                      b   rev                                         h   ui r
                 S.                                          h   an
                                                         S.a
                                                                                                             )
                                                                                                        ur
                                                                                                      (c
                                                                                              bum
                                                                                         lo
                                                                               u    rti                                    stn
                                                                          S .c                                           cha
                                                                                                  ajh                                    )
                                                                                                                                 i (j u z
                                                                                                                  epc      zuk i
                                                                                                        S   . juz
                                                                    ajh                                                                                                            0              0.2
                                                                 juz
                                                                  ajh


                                                         S. acaule (4x) wild species
                                                                                                                                                                                                                                     EVOLUTION




                                                                                                      Fig. 1.             (Figure continues on the opposite page.)



   The wild relatives of these landraces (Solanum section Petota) are                                                                              a monophyletic origin; (ii) the wild species progenitors were from
all tuber-bearing and include 190 wild species that are widely                                                                                     a group of very similar wild potato species classified in the Solanum
distributed in the Americas from the southwestern United States to                                                                                 brevicaule complex; and (iii) the landraces had their origin in the
southern Chile (17); they possess all ploidy levels of the cultivars, as                                                                           highlands of southern Peru.
well as hexaploids (2n 6x 72). Spooner et al. (18) studied the                                                                                        Although ploidy has been a major feature to define the
origin of the landraces S. tuberosum subsp. andigenum, S. tuberosum                                                                                cultivated species, many cultivated potato germplasm collections
subsp. tuberosum, S. phureja, and S. stenotomum with amplified                                                                                     lack chromosome numbers, and many assumptions of ploidy are
fragment length polymorphisms (AFLPs). They discovered that (i)                                                                                    likely in error. For example, Ghislain et al. (19) used simple
in contrast to all prior hypotheses, these species were shown to have                                                                              sequence repeats (SSRs) (also known as microsatellites) to

Spooner et al.                                                                                                                                                       PNAS          December 4, 2007     vol. 104    no. 49   19399
36


                                                                           48
                                                                      48

                      B


                                                                                                                                  48
                                                                                                                             48




                                                                                                            36 36 36
                                                                                                       36
                                                                                                            36 36           48      48
                                                                                                                                  48 48




                                                                                                                                            T
                                                                                                              48                       T
                                                                                                             48    48 48
                                                                                                               36 36



                                                                                                              24
                                                                                      24




                                              48


                                      X


                               Pink - Solanum ajanhuiri (ajh, 2x), S. juzepczukii (juz, 3x), S. curtilobum (cur, 5x)
                               Dark blue - S. tuberosum subsp. andigenum (adg, 4x)
                               Green - S. chaucha (cha, 3x)
                               Gray - S. tuberosum subsp. tuberosum (tub, 4x)
                               Red - S. phureja (phu, 2x)
                               Light blue - S. stenotomum (stn, 2x)
Fig. 1. Jaccard’s tree based on a dissimilarity matrix of 742 potato landraces and 8 wild species examined with 50 microsatellite primer pairs. (Inset) Overall
view of the entire tree. (A) Detail of the left-hand side of this tree, which contains mostly putative polyploid accessions (the ‘‘polyploid cluster’’) and accessions
of S. ajanhuiri, S. curtilobum, S. juzepczukii, and wild species. (B) Mostly putative diploid accessions and some triploids (the ‘‘diploid cluster’’). Chromosome
numbers (36 and 48) are labeled for accessions not 24 within the main red S. phureja cluster (in red) on the left right side of the tree or previously identified as
S. phureja but falling outside of the red cluster. The circled ‘‘T’’ (possessing a 241-bp deletion) and ‘‘X’’ (no deletion) designate accessions unexpected to occur
in these clusters because they either lack the T cytoplasm thought be largely confined to lowland Chile (area in the wedge) or lack this deletion (area outside
of the wedge containing mostly accessions from Venezuela to Argentina).


19400     www.pnas.org cgi doi 10.1073 pnas.0709796104                                                                                                  Spooner et al.
assess diversity in the S. phureja collection at the International    subsp. tuberosum accessions in the main cluster of this subspecies
Potato Center. Solanum phureja is widely grown in the Andes           (designated by the wedge in the polyploid cluster of Fig. 1)
from western Venezuela to central Bolivia and has been defined        possessed this deletion. Also in the area of the wedge are three
by short-day adaptation, diploid ploidy (2n 2x 24), and a lack        tetraploid accessions from Peru (dark blue); two of these possess
of tuber dormancy. SSR results, in combination with chromo-           the deletion, and one lacks it (the two accessions lacking the
some counts, uncovered fully 31% (32 of 102 accessions exam-          deletion are marked with ‘‘X’’).
ined) triploid and tetraploid accessions from the International          All four remaining accessions from Chile falling outside of this
Potato Center collection of S. phureja that were long assumed to      cluster (gray dots marked with ‘‘X’’) lack the 241-bp deletion
be exclusively diploid.                                               characteristic of this subspecies, suggesting misidentifications of
   The purpose of our study is to reexamine the support for           possible recent introductions of the S. tuberosum subsp. andige-
classification categories for landrace potatoes, using nuclear SSR    num into Chile. Thirteen of the 251 S. tuberosum subsp. andi-
markers developed for optimal utility in S. tuberosum regarding       genum accessions (5.2%; marked with ‘‘T’’ outside of the gray S.
polymorphism, quality scores, and genomic coverage (20), sup-         tuberosum subsp. tuberosum cluster) possessed the deletion,
plemented with a plastid DNA deletion marker as discussed             similar to the 4.4% reported in prior studies (above). These 13
below. Nuclear SSRs have been shown to be ideal markers for           accessions are widely distributed throughout the Andes in
detecting phylogenetically significant diversity within cultivated    Venezuela (2 accessions), Colombia (1 accession), Ecuador (13
potatoes (19, 21, 22). In addition, their codominant nature allows    accessions), Peru (4 accessions), Bolivia (2 accessions), and
them to identify polyploids when three or four bands (alleles) are    Argentina (1 accession). In addition, one of the S. stenotomum
found, as was shown by Ghislain et al. (19). This is particularly     accessions (putatively diploid) and two S. phureja accessions
important for our study, where few cultivated accessions have         (known as diploid) possessed this deletion, the first report of
been characterized for chromosome number yet ploidy has been          diploid potatoes possessing this deletion, because none of the
so important conceptually in defining the cultivated species.         accessions of S. stenotomum (215) and S. chaucha (150) previ-
Our study also used the 241-bp plastid deletion marker distin-        ously screened was found with this marker (19, 25). Unfortu-
guishing most populations of Chilean from Andean potato               nately, these three accessions do not have reliable collection
landraces (4, 12, 23).                                                information.
Results and Discussion                                                Reconsideration of the Classification of Cultivated Potato. In com-
SSR Neighbor-Joining (NJ) Tree. NJ results highlight a tendency to    bination with a recent morphological study (1), the SSR data
separate three broad groups: (i) putative diploids and triploids      support the reclassification of the cultivated potatoes into four
(all accessions in the diploid cluster Fig. 1), (ii) putative tet-    species: (i) S. tuberosum, (ii) S. ajanhuiri (diploid), (iii) S. juzepczukii
raploids and triploids (most accessions in the polyploid cluster of   (triploid), and (iv) S. curtilobum (pentaploid). We support dividing
Fig. 1), and (iii) the hybrid cultivated species S. ajanhuiri         S. tuberosum into two Cultivar Groups (Andigenum Group of
(diploid), S. juzepczukii (triploid), and S. curtilobum (penta-       upland Andean genotypes containing diploids, triploids, and tet-
ploid), grouped with the wild species. Bootstrap support above        raploids, and the Chilotanum Group of lowland tetraploid Chilean
50% is common in many small groups of species in the terminal         landraces). Because Cultivar Groups are taxonomic categories used
branches of the NJ tree (not shown because they greatly com-          to associate cultivated plants with traits that are of use to agricul-
plicate the graphic). However, bootstrap support above 50% is         turists, this classification is convenient to separate these populations
present only in the lower nodes supporting S. ajanhuiri, S.           that grow in different areas, are adapted to different day-length
juzepczukii, and S. curtilobum and the wild species.                  regimes, and have some degree of unilateral sexual incompatibility
   However, there are many exceptions of clustering by ploidy.        to the Andean populations. For the remaining ‘‘species’’ or Cultivar
Landraces of S. goniocalyx, as in the morphological study of          Groups, consistent and stable identifications are impossible, their
Huaman and Spooner (1), were invariably intermixed with
       ´                                                              classification as Linnean species is artificial, and their maintenance
those of S. stenotomum and are so labeled as this species on          as either species or Cultivar Groups only serves to perpetuate
Fig. 1. There are 28 putative triploid landraces of S. chaucha        confusion by breeders and gene bank managers, and the instability
present on the main polyploid cluster of the tree and 123 on          of names in the literature. For example, Ghislain et al. (19) showed
the main diploid cluster. There are 28 putative tetraploids (S.       S. phureja to be indefinable as traditionally recognized because
tuberosum subsp. andigenum and subsp. tuberosum) on the               prior authors incorrectly assumed that their assumption of diploidy
diploid cluster and 28 putative diploids (S. phureja and S.           was incorrect for 31% of the accessions, and our results showed
stenotomum) on the tetraploid cluster. In addition, S. phureja        many accessions of S. phureja to cluster with the polyploids. The
(the only cultivated species with extensive chromosome                recognition of S. phureja as either a species or Cultivar Group
counts) shows 18 of the 89 accessions to be triploid or               (Phureja Group), therefore, is no longer tenable because it is no
tetraploid. Regarding the S. phureja, Fig. 1 shows the position       longer diploid, does not exclusively possess low-dormancy tubers, is
of all accessions formerly identified as this species in the          not short-day adapted, and is not morphologically coherent (1). The
International Potato Center collection (24) but shown to be           other species (or Cultivar Groups) have ploidy as a major identi-
polyploid in the study of Ghislain et al. (19). Most are present      fying criterion. The results from S. phureja and this study indicate
                                                                                                                                                     EVOLUTION



in the main ‘‘S. phureja cluster’’ (red dots in the diploid cluster   that chromosome counts from other accessions of cultivated pota-
of Fig. 1), but this cluster also contains two accessions of S.       toes will uncover a high proportion of counts not matching expec-
tuberosum subsp. andigenum, two of S. stenotomum, and one of          tations based on their identifications.
S. chaucha, and with 10 accessions elsewhere on the diploid              Ploidy has been of great importance in the classification of
cluster and 16 on the polyploid cluster. As expected, most (22        cultivated potatoes, but our results show so many exceptions that it
of 27) of the S. tuberosum subsp. tuberosum accessions clus-          is a poor character to define gene pools. Cultivated potato fields
tered together (the area in the wedge in the polyploid cluster        contain mixtures of different ploidy levels (6, 26–32). Bukasov (33)
in Fig. 1). However, this cluster also contained three accessions     was the first to count chromosomes of the cultivated potatoes and
of S. tuberosum subsp. andigenum.                                     used ploidy variation to speculate on hybrid origins. The strong
                                                                      reliance on ploidy levels was clearly stated by Hawkes and Hjerting
241-bp Plastid Deletion. We determined the presence or absence        (34): ‘‘The chromosome number of 2n 36 largely helps to identify
of the 241-bp plastid deletion for all 742 cultivated accessions      S. chaucha, but morphological characters can also be used.’’
examined. As expected, most (22 of 23) of the S. tuberosum               Morphology is a poor character to define most species or Cultivar

Spooner et al.                                                                          PNAS     December 4, 2007     vol. 104    no. 49    19401
Groups except for the bitter potato species S. ajanhuiri, S. curti-               potato EST database at TIGR, and 23 from the University of Idaho
lobum, and S. juzepczukii. As shown by Huaman and Spooner (1),
                                                ´                                 (37). PCR reactions were performed in a 10- l volume containing
most traditionally recognized cultivated potato species have little               100 mM Tris HCl (Sigma), 20 mM (NH4)2SO4 (Merck), 2.5 mM
morphological support, and then only by using a suite of characters,              MgCl2 (Merck), 0.2 mM each dNTP (Amersham Biosciences), 0.3
all of which are shared with other taxa (polythetic support).                       M labeled M13 forward primer (LI-COR IRDye 700 or 800), 0.3
   The International Potato Center has collected cultivated pota-                   M M13-tailed SSR forward primer (Invitrogen), 0.2 M SSR
toes for 30 years and has invested tremendous effort in their                     reverse primer (Invitrogen), 1 unit of Taq polymerase (GIBCO/
identification. An examination of identification records at the                   BRL), and 15 ng of genomic DNA. PCR was carried out in a
International Potato Center shows many changes over the years,                    PTC-200 thermocycler (MJ Research). The program used was the
further showing the lack of stability of any character set to reliably            following: 4 min at 94°C, followed by 33 cycles of 50 sec at 94°C, 50
define most cultivated species.                                                   sec at annealing temperature (T°a), and 1 min at 72°C, then 4 min
   Potato gene banks are in great need of an integrated and                       at 72°C as a final extension step. PCR products were separated by
comprehensive program of ploidy determinations; controlled and                    electrophoresis on a LI-COR 4300 DNA analyzer system. The
replicated studies of tuber dormancy (which we suspect will high-                 molecular weight ladder was the LI-COR IRDye 50–350 bp size
light grades of dormancy, not the present/absent determinations                   standard and was loaded into gel each eight samples.
that exist today); photographically documented determinations of
tuber and flesh colors and tuber shapes; and determinations of                    SSR Allele Scoring. SSR alleles were detected and scored by using
tuber pigments, glycoalkaloid contents, carbohydrates, proteins,                  SAGA Generation 2 software (LI-COR). Size calibration and an
amino acids, minerals, and secondary metabolites, using functional                SSR ‘‘smiling line’’ were performed by using the molecular weight
genomics approaches, with all data publicly integrated into a readily             ladder (LI-COR IRDye 50–350). The SSR alleles were determined
searchable web-based bioinformatics database. Such a multicom-                    for size in bp of the upper band of the allele and scored as present
ponent system will serve the breeding community much better than                  (1) or absent (0). Missing data were scored as ‘‘9.’’
the outdated, unstable, and phylogenetically indefensible tradi-
tional classifications that exist today.                                          Data Analysis. Genetic analysis was performed by using the program
                                                                                  DARwin (38). A dissimilarity matrix was calculated by using
Materials and Methods                                                             Jaccard’s coefficient, 60% of minimal proportion of valid data
Plant Materials. A total of 742 potato landraces of all cultivated                required for each unit pair, and 500 replicate bootstrapping. The
potato species were examined: S. tuberosum subsp. andigenum,                      dendrogram was built by using the NJ method, using the seven wild
putatively tetraploid (251 accessions); S. ajanhuiri, diploid                     species accessions in the northern S. brevicaule complex as out-
(22); S. chaucha, triploid (151 accessions); S. tuberosum subsp.                  group. The NJ method developed by Saitou and Nei (39) estimates
tuberosum, tetraploid (27 accessions); S. curtilobum, penta-                      phylogenetic trees. Although based on the idea of parsimony (it
ploid (21 accessions); S. juzepczukii, triploid (35 accessions); S.               does yield relatively short estimated evolutionary trees), the NJ
phureja, diploid (104 accessions); S. stenotomum, diploid (131                    method does not attempt to obtain the shortest possible tree for a
accessions); 7 diploid wild species accessions in the northern                    set of data. Rather, it attempts to find a tree that is usually close to
S. brevicaule complex S. ambosinum Ochoa (1 accession), S.                        the true phylogenetic tree (40). This method allows the rooting of
bukasovii Juz. (4 accessions), and S. multiinterruptum Bitter (2                  trees on outgroups (in this case, the seven accessions of the S.
accessions); and the wild tetraploid species S. acaule Bitter (1                  brevicaule complex). The polymorphic information content (PIC)
accession) (750 accessions in total with the 8 wild species).                     was calculated as PIC 1          (pi2), where pi is the frequency of the
Selection of these wild species is based on recent amplified                      ith allele detected in all accessions (41). Data of somatic chromo-
fragment length polymorphism (AFLP) studies that docu-                            some counts for accessions of S. phureja were obtained from
mented the northern S. brevicaule complex wild species to be                      Ghislain et al. (19).
the progenitors of the cultivated potatoes and S. acaule
believed to be a wild species parent in the hybrid species S.                     Plastid DNA Polymorphism Detection. The 241-bp deletion was
juzepczukii and S. curtilobum. We qualify landrace collection                     analyzed for all 742 cultivated accessions by using the primers from
ploidy as ‘‘putative’’ because only S. phureja has been counted                   ref. 23. PCR amplification was performed in a volume of 10 l
in detail (19), that showed extensive examples of incorrect                       consisting of 18 ng of genomic DNA, 0.4 M each of primers
assumptions of ploidy as discussed above. Data of these                           (Invitrogen), 1 PCR buffer (PerkinElmer), 2.5 mM MgCl2
accessions that includes International Potato Center accession                    (PerkinElmer), 200 M each dNTP (Amersham Biosciences), and
number, taxonomic identification, ploidy when known, locality                     0.25 unit of Taq DNA polymerase (GIBCO/BRL). Thermal cycling
of collection, and average number of SSR alleles per accession                    was carried out in a PTC-200 thermocycler (MJ Research) (one
                                                                                  cycle of 4 min at 94°C, followed by 40 cycles of 45 sec at 94°C, 45
are available as a supporting information (SI) Dataset.
                                                                                  sec at 59°C, and 45 sec at 72°C, then terminated with one cycle of
                                                                                  4 min at 72°C). PCR products were separated by electrophoresis in
DNA Extraction, SSR Primers, PCR Conditions, and Electrophoresis.
                                                                                  a 1% agarose gel, and lambda phage digested by PstI was used as
Genomic DNA was obtained by using standard protocols at the
                                                                                  a molecular weight marker. The 241-bp plastid polymorphism was
International Potato Center (35). DNA concentration was calcu-
                                                                                  determined for size in bp and scored as ‘‘T’’ ( 200 bp for deleted
lated by using PicoGreen dsDNA quantitation reagent (Molecular
                                                                                  type) and ‘‘X’’ ( 440 bp for undeleted type).
Probes) and a TBS-380 Fluorometer (Turner BioSystems). DNA
dilutions were performed to achieve a final concentration of 3                    We thank David Douches and Lynn Bohs for comments on an earlier draft
ng/ l, using 96-well plates. We used 50 nuclear SSRs (see SI                      of the manuscript. This work was supported by the International Potato
Dataset) screened from 88 that included the 22 from the Potato                    Center, U.S. Department of Agriculture, Generation Challenge Program
Genetic Identity (PGI) kit (20), 13 from ESTs developed at the                    Grant SP1C2-2004-5, and National Science Foundation Grant DEB
Scottish Crop Research Institute (36), 30 identified by using the                 0316614 entitled ‘‘A world-wide treatment of Solanum’’ (to D.M.S.).


 1. Huaman Z, Spooner DM (2002) Am J Bot 89:947–965.
          ´                                                                        4.   Hosaka K (2003) Am J Potato Res 80:21–32.
 2. Hawkes JG (1990) The Potato: Evolution, Biodiversity, and Genetic Resources    5.   Spooner DM, Fajardo D, Bryan GJ (2007) Taxon 56:987–999.
    (Belhaven, London).                                                            6.   Johns T, Huaman Z, Ochoa CM, Schmiediche PE (1987) Syst Bot 12:541–552.
                                                                                                     ´
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19402     www.pnas.org cgi doi 10.1073 pnas.0709796104                                                                                            Spooner et al.
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                                                                                                                                                                          EVOLUTION




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Extensive simple sequence repeat genotyping of potato landraces supports a major reevaluation of their gene pool structure and classification

  • 1. Extensive simple sequence repeat genotyping of potato landraces supports a major reevaluation of their gene pool structure and classification David M. Spooner*†, Jorge Nunez‡, Guillermo Trujillo‡, Marıa del Rosario Herrera‡, Frank Guzman‡, and Marc Ghislain‡ ´˜ ´ ´ *Vegetable Crops Research Unit, Agricultural Research Service, U.S. Department of Agriculture, Department of Horticulture, University of Wisconsin, 1575 Linden Drive, Madison, WI 53706-1590; and ‡Applied Biotechnology Laboratory, International Potato Center, Apartado 1558, Lima 12, Peru Communicated by S. J. Peloquin, University of Wisconsin, Madison, WI, October 16, 2007 (received for review August 15, 2007) Contrasting taxonomic treatments of potato landraces have con- grown nearly as extensively as S. tuberosum, as outlined by tinued over the last century, with the recognition of anywhere Huaman and Spooner (1). ´ from 1 to 21 distinct Linnean species, or of Cultivar Groups within The relationships and extent of genetic differentiation be- the single species Solanum tuberosum. We provide one of the tween the Andean and Chilean landraces has long been contro- largest molecular marker studies of any crop landraces to date, to versial. Based on cytoplasmic sterility factors, geographical include an extensive study of 742 landraces of all cultivated species isolation, and ecological differences, Grun (9) suggested that (or Cultivar Groups) and 8 closely related wild species progenitors, Chilean landraces were distinct from Andean landraces. Hawkes with 50 nuclear simple sequence repeat (SSR) (also known as (2) distinguished the tetraploid Chilean from Andean landraces microsatellite) primer pairs and a plastid DNA deletion marker that by characters of the leaf and flower pedicel. Plastid restriction distinguishes most lowland Chilean from upland Andean land- site data documented five genotypes (A, C, S, T, and W types) races. Neighbor-joining results highlight a tendency to separate in the diploid and tetraploid Andean landraces, and the Chilean three groups: (i) putative diploids, (ii) putative tetraploids, and (iii) landraces had three types, A, T, and W (10, 11). The most the hybrid cultivated species S. ajanhuiri (diploid), S. juzepczukii frequently observed type in the Chilean landraces (21 of 24 or (triploid), and S. curtilobum (pentaploid). However, there are many 87.5% of the accessions examined) is type T, which is charac- exceptions to grouping by ploidy. Strong statistical support occurs terized by a 241-bp deletion (12). Conversely, 5 of the 113 (4.4%) only for S. ajanhuiri, S. juzepczukii, and S. curtilobum. In combina- accessions of S. tuberosum subsp. andigenum had the T type tion with recent morphological analyses and an examination of the (10–12). identification history of these collections, we support the reclas- Potato landraces have been classified into 21 species (13, 14), sification of the cultivated potatoes into four species: (i) S. tubero- 7 species with seven subspecies (2) and 9 species with two sum, with two Cultivar Groups (Andigenum Group of upland subspecies (15, 16), or as the single species S. tuberosum with 8 Andean genotypes containing diploids, triploids, and tetraploids, user-defined Cultivar Groups (1). Cultivar Groups are taxo- and the Chilotanum Group of lowland tetraploid Chilean land- nomic categories used by the International Code of Nomencla- races); (ii) S. ajanhuiri (diploid); (iii) S. juzepczukii (triploid); and (iv) ture of Cultivated Plants to associate cultivated plants with traits S. curtilobum (pentaploid). For other classifications, consistent and that are of use to agriculturists and are not meant to represent stable identifications are impossible, and their classification as natural groups or species in any classification philosophy. Ploidy species is artificial and only maintains the confusion of users of the levels in cultivated potatoes range from diploid (2n 2x 24), gene banks and literature. to triploid (2n 3x 36), to tetraploid (2n 4x 48), to pentaploid (2n 5x 60). Huaman and Spooner (1) examined ´ cultivated microsatellites sect. Petota Solanum tuberosum taxonomy the morphological support for the various classifications of potato landraces using representatives of all seven species from T he cultivated potato represents one of the most important food plants worldwide, yet interpretation of its gene pool structure remains controversial. Contrasting taxonomic treat- the classification of Hawkes (2). The results showed some morphological support for S. ajanhuiri, S. chaucha, S. curtilobum, and S. juzepczukii, lesser support for S. tuberosum subsp. tubero- ments of the landraces have continued over last century, with the sum, and no support for S. phureja and S. stenotomum. Whatever recognition of anywhere from 1 to 21 distinct Linnean species, morphological support for these entities was present was only by or of various Cultivar Groups within the single species S. using a suite of characters, all of which are shared with other taxa tuberosum (1). For consistency in usage in our article and to (polythetic support). These results, combined with their likely maintain the names most familiar to scientists, we use the seven hybrid origins, multiple origins, and evolutionary dynamics of species terminology of Hawkes (2). Indigenous cultivated (land- continuing hybridization, led Huaman and Spooner (1) to rec- ´ race) potatoes are widely distributed in the Andes from western ognize all landrace populations of cultivated potatoes as a single Venezuela, south to northern Argentina, and with another set of species, S. tuberosum, with the eight Cultivar Groups: Ajanhuiri landraces in south-central Chile in Chiloe Island and the adja- ´ Group, Andigenum Group, Chaucha Group, Chilotanum cent Chonos Archipelago. The Chilean landraces, although once Group, Curtilobum Group, Juzepczukii Group, Phureja Group, proposed to have arisen independently from central Chile (3), and Stenotomum Group (the latter containing all landraces of are secondarily derived from the Andean ones (2), likely after the Goniocalyx Group). hybridization with the Bolivian and Argentinean species Sola- num berthaultii (4), a species recently combined with the for- merly recognized wild species S. tarijense (5). Three of the Author contributions: J.N., G.T., M.d.R.H., F.G., and M.G. performed research; and D.M.S. Andean-cultivated species are hypothesized to be of hybrid wrote the paper. origins with cultivated potatoes and wild species: S. ajanhuiri [S. The authors declare no conflict of interest. stenotomum cultivated S. megistacrolobum wild (6)], S. juz- †To whom correspondence should be addressed. E-mail: david.spooner@ars.usda.gov. epczukii [S. stenotomum S. acaule wild (7, 8)], and S. curti- This article contains supporting information online at www.pnas.org/cgi/content/full/ lobum [S. andigenum cultivated S. juzepczukii (7, 8)]. The latter 0709796104/DC1. three ‘‘bitter potatoes’’ are grown in upland habitats and are not © 2007 by The National Academy of Sciences of the USA 19398 –19403 PNAS December 4, 2007 vol. 104 no. 49 www.pnas.org cgi doi 10.1073 pnas.0709796104
  • 2. 48 X T 48 48 48 48 48 A T T 48 phu 36 T cur 36 T T T T 48 48 X T 48 X T 48 T T X 63 All T except 52 2 as X X 75 89 100 X T p g rou ule jh) ic a i (a b rev h ui r S. h an S.a ) ur (c bum lo u rti stn S .c cha ajh ) i (j u z epc zuk i S . juz ajh 0 0.2 juz ajh S. acaule (4x) wild species EVOLUTION Fig. 1. (Figure continues on the opposite page.) The wild relatives of these landraces (Solanum section Petota) are a monophyletic origin; (ii) the wild species progenitors were from all tuber-bearing and include 190 wild species that are widely a group of very similar wild potato species classified in the Solanum distributed in the Americas from the southwestern United States to brevicaule complex; and (iii) the landraces had their origin in the southern Chile (17); they possess all ploidy levels of the cultivars, as highlands of southern Peru. well as hexaploids (2n 6x 72). Spooner et al. (18) studied the Although ploidy has been a major feature to define the origin of the landraces S. tuberosum subsp. andigenum, S. tuberosum cultivated species, many cultivated potato germplasm collections subsp. tuberosum, S. phureja, and S. stenotomum with amplified lack chromosome numbers, and many assumptions of ploidy are fragment length polymorphisms (AFLPs). They discovered that (i) likely in error. For example, Ghislain et al. (19) used simple in contrast to all prior hypotheses, these species were shown to have sequence repeats (SSRs) (also known as microsatellites) to Spooner et al. PNAS December 4, 2007 vol. 104 no. 49 19399
  • 3. 36 48 48 B 48 48 36 36 36 36 36 36 48 48 48 48 T 48 T 48 48 48 36 36 24 24 48 X Pink - Solanum ajanhuiri (ajh, 2x), S. juzepczukii (juz, 3x), S. curtilobum (cur, 5x) Dark blue - S. tuberosum subsp. andigenum (adg, 4x) Green - S. chaucha (cha, 3x) Gray - S. tuberosum subsp. tuberosum (tub, 4x) Red - S. phureja (phu, 2x) Light blue - S. stenotomum (stn, 2x) Fig. 1. Jaccard’s tree based on a dissimilarity matrix of 742 potato landraces and 8 wild species examined with 50 microsatellite primer pairs. (Inset) Overall view of the entire tree. (A) Detail of the left-hand side of this tree, which contains mostly putative polyploid accessions (the ‘‘polyploid cluster’’) and accessions of S. ajanhuiri, S. curtilobum, S. juzepczukii, and wild species. (B) Mostly putative diploid accessions and some triploids (the ‘‘diploid cluster’’). Chromosome numbers (36 and 48) are labeled for accessions not 24 within the main red S. phureja cluster (in red) on the left right side of the tree or previously identified as S. phureja but falling outside of the red cluster. The circled ‘‘T’’ (possessing a 241-bp deletion) and ‘‘X’’ (no deletion) designate accessions unexpected to occur in these clusters because they either lack the T cytoplasm thought be largely confined to lowland Chile (area in the wedge) or lack this deletion (area outside of the wedge containing mostly accessions from Venezuela to Argentina). 19400 www.pnas.org cgi doi 10.1073 pnas.0709796104 Spooner et al.
  • 4. assess diversity in the S. phureja collection at the International subsp. tuberosum accessions in the main cluster of this subspecies Potato Center. Solanum phureja is widely grown in the Andes (designated by the wedge in the polyploid cluster of Fig. 1) from western Venezuela to central Bolivia and has been defined possessed this deletion. Also in the area of the wedge are three by short-day adaptation, diploid ploidy (2n 2x 24), and a lack tetraploid accessions from Peru (dark blue); two of these possess of tuber dormancy. SSR results, in combination with chromo- the deletion, and one lacks it (the two accessions lacking the some counts, uncovered fully 31% (32 of 102 accessions exam- deletion are marked with ‘‘X’’). ined) triploid and tetraploid accessions from the International All four remaining accessions from Chile falling outside of this Potato Center collection of S. phureja that were long assumed to cluster (gray dots marked with ‘‘X’’) lack the 241-bp deletion be exclusively diploid. characteristic of this subspecies, suggesting misidentifications of The purpose of our study is to reexamine the support for possible recent introductions of the S. tuberosum subsp. andige- classification categories for landrace potatoes, using nuclear SSR num into Chile. Thirteen of the 251 S. tuberosum subsp. andi- markers developed for optimal utility in S. tuberosum regarding genum accessions (5.2%; marked with ‘‘T’’ outside of the gray S. polymorphism, quality scores, and genomic coverage (20), sup- tuberosum subsp. tuberosum cluster) possessed the deletion, plemented with a plastid DNA deletion marker as discussed similar to the 4.4% reported in prior studies (above). These 13 below. Nuclear SSRs have been shown to be ideal markers for accessions are widely distributed throughout the Andes in detecting phylogenetically significant diversity within cultivated Venezuela (2 accessions), Colombia (1 accession), Ecuador (13 potatoes (19, 21, 22). In addition, their codominant nature allows accessions), Peru (4 accessions), Bolivia (2 accessions), and them to identify polyploids when three or four bands (alleles) are Argentina (1 accession). In addition, one of the S. stenotomum found, as was shown by Ghislain et al. (19). This is particularly accessions (putatively diploid) and two S. phureja accessions important for our study, where few cultivated accessions have (known as diploid) possessed this deletion, the first report of been characterized for chromosome number yet ploidy has been diploid potatoes possessing this deletion, because none of the so important conceptually in defining the cultivated species. accessions of S. stenotomum (215) and S. chaucha (150) previ- Our study also used the 241-bp plastid deletion marker distin- ously screened was found with this marker (19, 25). Unfortu- guishing most populations of Chilean from Andean potato nately, these three accessions do not have reliable collection landraces (4, 12, 23). information. Results and Discussion Reconsideration of the Classification of Cultivated Potato. In com- SSR Neighbor-Joining (NJ) Tree. NJ results highlight a tendency to bination with a recent morphological study (1), the SSR data separate three broad groups: (i) putative diploids and triploids support the reclassification of the cultivated potatoes into four (all accessions in the diploid cluster Fig. 1), (ii) putative tet- species: (i) S. tuberosum, (ii) S. ajanhuiri (diploid), (iii) S. juzepczukii raploids and triploids (most accessions in the polyploid cluster of (triploid), and (iv) S. curtilobum (pentaploid). We support dividing Fig. 1), and (iii) the hybrid cultivated species S. ajanhuiri S. tuberosum into two Cultivar Groups (Andigenum Group of (diploid), S. juzepczukii (triploid), and S. curtilobum (penta- upland Andean genotypes containing diploids, triploids, and tet- ploid), grouped with the wild species. Bootstrap support above raploids, and the Chilotanum Group of lowland tetraploid Chilean 50% is common in many small groups of species in the terminal landraces). Because Cultivar Groups are taxonomic categories used branches of the NJ tree (not shown because they greatly com- to associate cultivated plants with traits that are of use to agricul- plicate the graphic). However, bootstrap support above 50% is turists, this classification is convenient to separate these populations present only in the lower nodes supporting S. ajanhuiri, S. that grow in different areas, are adapted to different day-length juzepczukii, and S. curtilobum and the wild species. regimes, and have some degree of unilateral sexual incompatibility However, there are many exceptions of clustering by ploidy. to the Andean populations. For the remaining ‘‘species’’ or Cultivar Landraces of S. goniocalyx, as in the morphological study of Groups, consistent and stable identifications are impossible, their Huaman and Spooner (1), were invariably intermixed with ´ classification as Linnean species is artificial, and their maintenance those of S. stenotomum and are so labeled as this species on as either species or Cultivar Groups only serves to perpetuate Fig. 1. There are 28 putative triploid landraces of S. chaucha confusion by breeders and gene bank managers, and the instability present on the main polyploid cluster of the tree and 123 on of names in the literature. For example, Ghislain et al. (19) showed the main diploid cluster. There are 28 putative tetraploids (S. S. phureja to be indefinable as traditionally recognized because tuberosum subsp. andigenum and subsp. tuberosum) on the prior authors incorrectly assumed that their assumption of diploidy diploid cluster and 28 putative diploids (S. phureja and S. was incorrect for 31% of the accessions, and our results showed stenotomum) on the tetraploid cluster. In addition, S. phureja many accessions of S. phureja to cluster with the polyploids. The (the only cultivated species with extensive chromosome recognition of S. phureja as either a species or Cultivar Group counts) shows 18 of the 89 accessions to be triploid or (Phureja Group), therefore, is no longer tenable because it is no tetraploid. Regarding the S. phureja, Fig. 1 shows the position longer diploid, does not exclusively possess low-dormancy tubers, is of all accessions formerly identified as this species in the not short-day adapted, and is not morphologically coherent (1). The International Potato Center collection (24) but shown to be other species (or Cultivar Groups) have ploidy as a major identi- polyploid in the study of Ghislain et al. (19). Most are present fying criterion. The results from S. phureja and this study indicate EVOLUTION in the main ‘‘S. phureja cluster’’ (red dots in the diploid cluster that chromosome counts from other accessions of cultivated pota- of Fig. 1), but this cluster also contains two accessions of S. toes will uncover a high proportion of counts not matching expec- tuberosum subsp. andigenum, two of S. stenotomum, and one of tations based on their identifications. S. chaucha, and with 10 accessions elsewhere on the diploid Ploidy has been of great importance in the classification of cluster and 16 on the polyploid cluster. As expected, most (22 cultivated potatoes, but our results show so many exceptions that it of 27) of the S. tuberosum subsp. tuberosum accessions clus- is a poor character to define gene pools. Cultivated potato fields tered together (the area in the wedge in the polyploid cluster contain mixtures of different ploidy levels (6, 26–32). Bukasov (33) in Fig. 1). However, this cluster also contained three accessions was the first to count chromosomes of the cultivated potatoes and of S. tuberosum subsp. andigenum. used ploidy variation to speculate on hybrid origins. The strong reliance on ploidy levels was clearly stated by Hawkes and Hjerting 241-bp Plastid Deletion. We determined the presence or absence (34): ‘‘The chromosome number of 2n 36 largely helps to identify of the 241-bp plastid deletion for all 742 cultivated accessions S. chaucha, but morphological characters can also be used.’’ examined. As expected, most (22 of 23) of the S. tuberosum Morphology is a poor character to define most species or Cultivar Spooner et al. PNAS December 4, 2007 vol. 104 no. 49 19401
  • 5. Groups except for the bitter potato species S. ajanhuiri, S. curti- potato EST database at TIGR, and 23 from the University of Idaho lobum, and S. juzepczukii. As shown by Huaman and Spooner (1), ´ (37). PCR reactions were performed in a 10- l volume containing most traditionally recognized cultivated potato species have little 100 mM Tris HCl (Sigma), 20 mM (NH4)2SO4 (Merck), 2.5 mM morphological support, and then only by using a suite of characters, MgCl2 (Merck), 0.2 mM each dNTP (Amersham Biosciences), 0.3 all of which are shared with other taxa (polythetic support). M labeled M13 forward primer (LI-COR IRDye 700 or 800), 0.3 The International Potato Center has collected cultivated pota- M M13-tailed SSR forward primer (Invitrogen), 0.2 M SSR toes for 30 years and has invested tremendous effort in their reverse primer (Invitrogen), 1 unit of Taq polymerase (GIBCO/ identification. An examination of identification records at the BRL), and 15 ng of genomic DNA. PCR was carried out in a International Potato Center shows many changes over the years, PTC-200 thermocycler (MJ Research). The program used was the further showing the lack of stability of any character set to reliably following: 4 min at 94°C, followed by 33 cycles of 50 sec at 94°C, 50 define most cultivated species. sec at annealing temperature (T°a), and 1 min at 72°C, then 4 min Potato gene banks are in great need of an integrated and at 72°C as a final extension step. PCR products were separated by comprehensive program of ploidy determinations; controlled and electrophoresis on a LI-COR 4300 DNA analyzer system. The replicated studies of tuber dormancy (which we suspect will high- molecular weight ladder was the LI-COR IRDye 50–350 bp size light grades of dormancy, not the present/absent determinations standard and was loaded into gel each eight samples. that exist today); photographically documented determinations of tuber and flesh colors and tuber shapes; and determinations of SSR Allele Scoring. SSR alleles were detected and scored by using tuber pigments, glycoalkaloid contents, carbohydrates, proteins, SAGA Generation 2 software (LI-COR). Size calibration and an amino acids, minerals, and secondary metabolites, using functional SSR ‘‘smiling line’’ were performed by using the molecular weight genomics approaches, with all data publicly integrated into a readily ladder (LI-COR IRDye 50–350). The SSR alleles were determined searchable web-based bioinformatics database. Such a multicom- for size in bp of the upper band of the allele and scored as present ponent system will serve the breeding community much better than (1) or absent (0). Missing data were scored as ‘‘9.’’ the outdated, unstable, and phylogenetically indefensible tradi- tional classifications that exist today. Data Analysis. Genetic analysis was performed by using the program DARwin (38). A dissimilarity matrix was calculated by using Materials and Methods Jaccard’s coefficient, 60% of minimal proportion of valid data Plant Materials. A total of 742 potato landraces of all cultivated required for each unit pair, and 500 replicate bootstrapping. The potato species were examined: S. tuberosum subsp. andigenum, dendrogram was built by using the NJ method, using the seven wild putatively tetraploid (251 accessions); S. ajanhuiri, diploid species accessions in the northern S. brevicaule complex as out- (22); S. chaucha, triploid (151 accessions); S. tuberosum subsp. group. The NJ method developed by Saitou and Nei (39) estimates tuberosum, tetraploid (27 accessions); S. curtilobum, penta- phylogenetic trees. Although based on the idea of parsimony (it ploid (21 accessions); S. juzepczukii, triploid (35 accessions); S. does yield relatively short estimated evolutionary trees), the NJ phureja, diploid (104 accessions); S. stenotomum, diploid (131 method does not attempt to obtain the shortest possible tree for a accessions); 7 diploid wild species accessions in the northern set of data. Rather, it attempts to find a tree that is usually close to S. brevicaule complex S. ambosinum Ochoa (1 accession), S. the true phylogenetic tree (40). This method allows the rooting of bukasovii Juz. (4 accessions), and S. multiinterruptum Bitter (2 trees on outgroups (in this case, the seven accessions of the S. accessions); and the wild tetraploid species S. acaule Bitter (1 brevicaule complex). The polymorphic information content (PIC) accession) (750 accessions in total with the 8 wild species). was calculated as PIC 1 (pi2), where pi is the frequency of the Selection of these wild species is based on recent amplified ith allele detected in all accessions (41). Data of somatic chromo- fragment length polymorphism (AFLP) studies that docu- some counts for accessions of S. phureja were obtained from mented the northern S. brevicaule complex wild species to be Ghislain et al. (19). the progenitors of the cultivated potatoes and S. acaule believed to be a wild species parent in the hybrid species S. Plastid DNA Polymorphism Detection. The 241-bp deletion was juzepczukii and S. curtilobum. We qualify landrace collection analyzed for all 742 cultivated accessions by using the primers from ploidy as ‘‘putative’’ because only S. phureja has been counted ref. 23. PCR amplification was performed in a volume of 10 l in detail (19), that showed extensive examples of incorrect consisting of 18 ng of genomic DNA, 0.4 M each of primers assumptions of ploidy as discussed above. Data of these (Invitrogen), 1 PCR buffer (PerkinElmer), 2.5 mM MgCl2 accessions that includes International Potato Center accession (PerkinElmer), 200 M each dNTP (Amersham Biosciences), and number, taxonomic identification, ploidy when known, locality 0.25 unit of Taq DNA polymerase (GIBCO/BRL). Thermal cycling of collection, and average number of SSR alleles per accession was carried out in a PTC-200 thermocycler (MJ Research) (one cycle of 4 min at 94°C, followed by 40 cycles of 45 sec at 94°C, 45 are available as a supporting information (SI) Dataset. sec at 59°C, and 45 sec at 72°C, then terminated with one cycle of 4 min at 72°C). PCR products were separated by electrophoresis in DNA Extraction, SSR Primers, PCR Conditions, and Electrophoresis. a 1% agarose gel, and lambda phage digested by PstI was used as Genomic DNA was obtained by using standard protocols at the a molecular weight marker. The 241-bp plastid polymorphism was International Potato Center (35). DNA concentration was calcu- determined for size in bp and scored as ‘‘T’’ ( 200 bp for deleted lated by using PicoGreen dsDNA quantitation reagent (Molecular type) and ‘‘X’’ ( 440 bp for undeleted type). Probes) and a TBS-380 Fluorometer (Turner BioSystems). DNA dilutions were performed to achieve a final concentration of 3 We thank David Douches and Lynn Bohs for comments on an earlier draft ng/ l, using 96-well plates. We used 50 nuclear SSRs (see SI of the manuscript. This work was supported by the International Potato Dataset) screened from 88 that included the 22 from the Potato Center, U.S. Department of Agriculture, Generation Challenge Program Genetic Identity (PGI) kit (20), 13 from ESTs developed at the Grant SP1C2-2004-5, and National Science Foundation Grant DEB Scottish Crop Research Institute (36), 30 identified by using the 0316614 entitled ‘‘A world-wide treatment of Solanum’’ (to D.M.S.). 1. Huaman Z, Spooner DM (2002) Am J Bot 89:947–965. ´ 4. Hosaka K (2003) Am J Potato Res 80:21–32. 2. Hawkes JG (1990) The Potato: Evolution, Biodiversity, and Genetic Resources 5. Spooner DM, Fajardo D, Bryan GJ (2007) Taxon 56:987–999. (Belhaven, London). 6. Johns T, Huaman Z, Ochoa CM, Schmiediche PE (1987) Syst Bot 12:541–552. ´ 3. Ugent D, Dillehay T, Ramirez C (1987) Econ Bot 41:17–27. 7. Hawkes JG (1962) Z Pflanzenzuecht 47:1–14. 19402 www.pnas.org cgi doi 10.1073 pnas.0709796104 Spooner et al.
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