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RESEARCH ARTICLE Genetic relationships in Cucurbita pepo (pumpkin, squash, gourd) as viewed with high frequency oligonucleotide– targeting active gene (HFO–TAG) markers Harry S. Paris Adi Doron-Faigenboim Umesh K. Reddy Ryan Donahoo Amnon Levi Received: 6 October 2014 / Accepted: 12 January 2015 Ó Springer Science+Business Media Dordrecht 2015 Abstract Cucurbita pepo is a highly diverse, eco- nomically important member of the Cucurbitaceae. C. pepo encompasses hundreds of cultivars of pump- kins, squash, and gourds. Although C. pepo has been scrutinized with various types of DNA markers, the relationships among the cultivar-groups of C. pepo subsp. pepo, the more widely grown subspecies, have not heretofore been adequately resolved. We assessed genetic relationships among 68 accessions of Cucur- bita pepo, including 48 from C. pepo subsp. pepo, using polymorphisms in 539 high frequency oligonu- cleotide–targeting active gene (HFO–TAG) frag- ments, that preferably represent coding regions of the genome. Dissimilarities among accessions were calculated, a dendrogram was constructed, and prin- cipal component analyses were conducted. Dissimilarities demarcated the four edible-fruited cultivar-groups of C. pepo subsp. pepo, Cocozelle, Pumpkin, Vegetable Marrow, and Zucchini. Further- more, the results indicate that the Old World pumpkins as well as the long-fruited cultivar-groups of C. pepo subsp. pepo (cocozelle, vegetable marrow, and zuc- chini) evolved from spontaneous crossing and gene exchange between pumpkins derived from northern North America and pumpkins derived from southern North America. Consistent with pictorial and narrative historical records, such crossing appears to have occurred in Renaissance Europe within the first decades of the European contact with North America. The Old World pumpkins are more closely related to the long-fruited cultivar-groups than are the native North American pumpkins. Keywords Crop evolution Cucurbita pepo subsp. pepo Cucurbitaceae Nuclear DNA markers Plant breeding Pumpkin Squash Zucchini Introduction Cucurbita pepo L. (2n = 2x = 40) is a highly diverse, economically important crop species encompassing summer squash as well as many pumpkins, winter squash, and ornamental gourds (Paris et al. 2012). This species is native to North America and is thought to have been domesticated first in southern Mexico H. S. Paris (&) Newe Ya‘ar Research Center, Agricultural Research Organization, P. O. Box 1021, 30-095 Ramat Yishay, Israel e-mail: [email protected] A. Doron-Faigenboim Volcani Center, Agricultural Research Organization, P. O. Box 6, 50-250 Bet Dagan, Israel U. K. Reddy Department of Biology, West Virginia State University, Institute, WV 25112-1000, USA R. Donahoo A. Levi U.S. Vegetable Laboratory, USDA, ARS, 2700 Savannah Highway, Charleston, SC 29414, USA 123 Genet Resour Crop Evol DOI 10.1007/s10722-015-0218-6
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Genetic relationships in Cucurbita pepo (pumpkin, squash, gourd) as viewed with high frequency oligonucleotide–targeting active gene (HFO–TAG) markers

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Page 1: Genetic relationships in Cucurbita pepo (pumpkin, squash, gourd) as viewed with high frequency oligonucleotide–targeting active gene (HFO–TAG) markers

RESEARCH ARTICLE

Genetic relationships in Cucurbita pepo (pumpkin, squash,gourd) as viewed with high frequency oligonucleotide–targeting active gene (HFO–TAG) markers

Harry S. Paris • Adi Doron-Faigenboim •

Umesh K. Reddy • Ryan Donahoo • Amnon Levi

Received: 6 October 2014 / Accepted: 12 January 2015

� Springer Science+Business Media Dordrecht 2015

Abstract Cucurbita pepo is a highly diverse, eco-

nomically important member of the Cucurbitaceae.

C. pepo encompasses hundreds of cultivars of pump-

kins, squash, and gourds. Although C. pepo has been

scrutinized with various types of DNA markers, the

relationships among the cultivar-groups of C. pepo

subsp. pepo, the more widely grown subspecies, have

not heretofore been adequately resolved. We assessed

genetic relationships among 68 accessions of Cucur-

bita pepo, including 48 from C. pepo subsp. pepo,

using polymorphisms in 539 high frequency oligonu-

cleotide–targeting active gene (HFO–TAG) frag-

ments, that preferably represent coding regions of

the genome. Dissimilarities among accessions were

calculated, a dendrogram was constructed, and prin-

cipal component analyses were conducted.

Dissimilarities demarcated the four edible-fruited

cultivar-groups of C. pepo subsp. pepo, Cocozelle,

Pumpkin, Vegetable Marrow, and Zucchini. Further-

more, the results indicate that the Old World pumpkins

as well as the long-fruited cultivar-groups of C. pepo

subsp. pepo (cocozelle, vegetable marrow, and zuc-

chini) evolved from spontaneous crossing and gene

exchange between pumpkins derived from northern

North America and pumpkins derived from southern

North America. Consistent with pictorial and narrative

historical records, such crossing appears to have

occurred in Renaissance Europe within the first

decades of the European contact with North America.

The Old World pumpkins are more closely related to

the long-fruited cultivar-groups than are the native

North American pumpkins.

Keywords Crop evolution � Cucurbita pepo subsp.

pepo � Cucurbitaceae � Nuclear DNA markers � Plant

breeding � Pumpkin � Squash � Zucchini

Introduction

Cucurbita pepo L. (2n = 2x = 40) is a highly diverse,

economically important crop species encompassing

summer squash as well as many pumpkins, winter

squash, and ornamental gourds (Paris et al. 2012). This

species is native to North America and is thought to

have been domesticated first in southern Mexico

H. S. Paris (&)

Newe Ya‘ar Research Center, Agricultural Research

Organization, P. O. Box 1021, 30-095 Ramat Yishay,

Israel

e-mail: [email protected]

A. Doron-Faigenboim

Volcani Center, Agricultural Research Organization,

P. O. Box 6, 50-250 Bet Dagan, Israel

U. K. Reddy

Department of Biology, West Virginia State University,

Institute, WV 25112-1000, USA

R. Donahoo � A. Levi

U.S. Vegetable Laboratory, USDA, ARS, 2700 Savannah

Highway, Charleston, SC 29414, USA

123

Genet Resour Crop Evol

DOI 10.1007/s10722-015-0218-6

Page 2: Genetic relationships in Cucurbita pepo (pumpkin, squash, gourd) as viewed with high frequency oligonucleotide–targeting active gene (HFO–TAG) markers

approximately 10,000 years ago (Whitaker and Cutler

1971, 1986; Lira and Montes 1994; Smith 1997).

Considerable evidence places a subsequent, indepen-

dent domestication in the eastern or central United

States about 5,000 years ago (Whitaker and Carter

1946; Smith 2006). These domestication events,

geographically quite distant, must have occurred in

genetically distinct populations. Allozyme variation

and DNA sequence polymorphisms have revealed a

strong dichotomy in cultivated C. pepo (Decker-

Walters 1990). Descendants of the older, more south-

erly domestication have been given subspecific status

as C. pepo subsp. pepo. However, wild populations of

subsp. pepo have not been described (Nee 1990).

Descendants of the more recent and northerly domes-

tication have been given subspecific status as C. pepo

subsp. texana (Scheele) Filov. Wild populations of

subsp. texana have been described from Texas and

much of the southeastern and central United States

(Erwin 1931; Bailey 1943; Decker and Wilson 1987;

Cowan 1997). Most cultigens belong to the former

subspecies but many others belong to the latter (Paris

et al. 2006, 2012). Wild populations of a third lineage

occur in a geographically intermediate region, the

states of Tamaulipas and Nuevo Leon in northeastern

Mexico (Bailey 1943; Andres 1987; Nee 1990). They

have a separate taxonomic designation, C. pepo subsp.

fraterna (Bailey) Lira, Andres et Nee, but as far as is

known have no domesticated descendants.

Cucurbita pepo, under thousands of years of

cultivation, has diversified greatly into an astonishing

range of fruit sizes, shapes, topographies, and colors

(Duchesne 1786; Paris 2007). Much of the stark

diversity in fruit shape has been driven by differing

uses of the fruits as food, for consumption of whole

immature fruits or of mature fruit flesh or seeds (Paris

et al. 2012). Differences in fruit shape, a polygenic

characteristic (Emerson 1910; Sinnott 1935) form the

basis of a horticultural classification recognizing eight

groups of edible-fruited cultivars (Paris 1986).

Accordingly, these are Acorn (turbinate, furrowed),

Scallop (flat, lobed), Crookneck (long, narrow

necked), Straightneck (long, with proximal constric-

tion), Pumpkin (round or nearly round), Vegetable

Marrow (short, tapered cylindrical), Cocozelle (long,

bulbous cylindrical) and Zucchini (uniformly cylin-

drical). The first four cultivar-groups are in C. pepo

subsp. texana and the last four are in C. pepo subsp.

pepo (Paris 2000). Both subspecies also encompass

various cultivars of small-fruited, ornamental gourds,

those of the former subspecies can be referred to as

‘‘ovifera gourds’’ and those of the latter as ‘‘pepo

gourds’’.

Various nuclear DNA-sequence polymorphisms

have been employed to examine genetic relationships

among Cucurbita pepo accessions with ever-increas-

ing precision (Lebeda et al. 2007). The markers used

have included random amplified polymorphic DNAs

(RAPDs), amplified fragment length polymorphisms

(AFLPs), inter-simple sequence repeats (ISSRs), sim-

ple sequence repeats (SSRs), and sequence-related

amplified polymorphisms (SRAPs). The results of

these investigations have helped clarify a number of

genetic relationships within the species, most notably

differentiating the subspecies of C. pepo and their

constituent cultivar-groups. Using the various mark-

ers, accessions within each of the four edible-fruited

cultivar-groups (fruit shapes) of C. pepo subsp. texana

were clearly seen to be associated with one another. Of

the four edible-fruited cultivar-groups of C. pepo

subsp. pepo, only the Zucchini Group (cultivars with

uniformly cylindrical-shaped fruits) was as clearly

defined, the other three being less clearly separated

from one another (Paris et al. 2003; Gong et al. 2012).

Recently, a new type of polymerase chain reaction

(PCR) marker named ‘‘high frequency oligonucleo-

tide–targeting active genes (HFO–TAG)’’ was devel-

oped (Levi et al. 2010). HFO–TAG markers are

dominant, produced by primers representing oligonu-

cleotides that exist in high frequency in expressed

sequence-tag (EST) unigenes, and are highly repro-

ducible and polymorphic. Therefore, HFO–TAGs

could be expected to depict genetic relationships more

in accordance with phenotypic variability than DNA

markers that represent non-coding regions. Indeed,

HFO–TAGs have resulted in a much improved

assessment of genetic diversity within Citrullus

(watermelon and colocynth) (Levi et al. 2013).

The present work focused mainly on Cucurbita

pepo subsp. pepo because relationships among culti-

var-groups of this subspecies have not been as well

resolved as in C. pepo subsp. texana. The objective

here is to obtain an improved definition of the elements

within this subspecies and to increase understanding of

the evolution and interrelationships of its cultivar-

groups by employing HFO–TAG markers.

Genet Resour Crop Evol

123

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Materials and methods

Plant material

Sixty-eight accessions of Cucurbita pepo were chosen

for study, most of which are of C. pepo subsp. pepo

(Table 1). Some of the subsp. pepo accessions have

been examined with other DNA markers (Paris et al.

2003; Gong et al. 2012) and are included here for

comparative purposes. Others, some of which exhibit

intriguing combinations of traits, have not previously

been subjected to such scrutiny. Four accessions of

Citrullus Schrad. ex Eckl. et Zeyh. and two of

Lagenaria Ser. were included as checks and to obtain

a picture of phylogenetic relationships among these

three cultivated genera of the Cucurbitaceae.

Each of the 68 accessions of Cucurbita pepo was

assigned a tripartite abbreviation (Table 1). The first part

is formed by one letter designating subspecies (P = pepo,

T = texana, F = fraterna, and accessions of question-

able or uncertain subspecific affiliation were designated as

Q = questionable and U = uncertain) (Paris et al. 2003;

Gong et al. 2012). The second part is formed by a two-

letter abbreviation designatingcultivar-grouporaffiliation

(for example, PU = Pumpkin, CO = Cocozelle,

SN = Straightneck, GO = Ovifera Gourd, GT = Wild

texana Gourd, ZU = Zucchini). The third part is formed

by a three-letter abbreviation of the name of the accession

(for example, CTF = ‘Connecticut Field’, SFF = ‘Saf-

fron’, TRF = ‘True French’). In such fashion, the subsp.

pepo Pumpkin ‘Connecticut Field’ was abbreviated

P-PU-CTF, the subsp. texana Straightneck squash ‘Saf-

fron’ was abbreviated T-SN-SFF, and the subsp. pepo

Zucchini ‘True French’ was abbreviated P-ZU-TRF.

One mature fruit from many of the accessions is

depicted in Fig. 1 and others are depicted elsewhere

(Paris 2001; Paris and Nerson 2003; Goldman 2004;

Paris et al. 2012). Seeds of each accession of Cucurbita

pepo were derived from the collection maintained at the

Agricultural Research Organization, Newe Ya‘ar

Research Center (Paris 2001). The original seed samples

were obtained frompublic and private sources and nearly

all of them are derived from North America or Europe

(Table 1). The identity of each accession was checked

and verified by sowing seeds and growing out at least

eight plants in the field. Plant growth habit was scored

from field observation as bush, open bush, or vine.

Most of the seeds used for producing plants for

DNA extraction were not from the original seed

samples but instead derived from seeds obtained by

self- or sib-pollinations at Newe Ya‘ar (Table 1). A

few of the original seed samples produced plants that

had noticeable variation among one another in vege-

tative and reproductive characteristics; in such cases

the plants selected for self- or sib-pollination were

those that were most representative of the accession,

based on its name, catalogue description, or the

majority of the plants observed.

The four Citrullus and two Lagenaria accessions

were also assigned tripartite abbreviations, beginning

with C for the former and L for the latter. The four

accessions of Citrullus used for this study were the

dessert watermelons, C. lanatus (Thunb.) Matsum. et

Nakai ‘Black Diamond’ (C-LA-BDI) and ‘Charleston

Gray’ (C-LA-CHG), the citron watermelon, C. lanatus

subsp. lanatus syn. C. amarus Schrad. PI 296341 (C-

LA-341), and the colocynth, C. colocynthis (L.)

Schrad. PI 386015 (C-CC-015). The two accessions

of Lagenaria were L. siceraria (Mol.) Standl. PI

271477 (L-SI-477) and PI 442369 (L-SI-369). The

seeds of these accessions of Citrullus and Lagenaria

are maintained at the U.S. Vegetable Laboratory,

A.R.S./U.S.D.A., Charleston, SC USA.

DNA extraction

Young leaves were collected from three or four two-

week-old plants of each accession and stored at

-80 �C for later DNA isolation. The DNA was

isolated from the frozen leaves using the method

described by Levi and Thomas (1999).

PCR amplification and analysis using HFO–TAG

primers

The identification and development of HFO–TAG

primers using expressed-sequence-tag (EST) data of

watermelon fruit have been described by Levi et al.

(2010). Twenty-two of these HFO–TAG primers

(Table 2) were used in this study.

DNA amplification conditions and analysis

The PCR reaction cocktail (25 lL) contained 20 lM

NaCl, 50 mM Tris–HCl pH 9, 0.1 % Triton-X-100,

0.01 % gelatin, 1.6 mM MgCl2, 200 lM each of

dNTPs (Sigma-Aldrich, St. Louis, MO), 100 lM

primer, five units GoTaq� DNA polymerase

Genet Resour Crop Evol

123

Page 4: Genetic relationships in Cucurbita pepo (pumpkin, squash, gourd) as viewed with high frequency oligonucleotide–targeting active gene (HFO–TAG) markers

Table 1 Classification and sources of seeds of 68 Cucurbita pepo accessions

Accession name Accession

abbreviation

Accession pedigreea Origin (seed source) Plant growth

habit

Royal Acorn T-AC-RAC RACc-S USA (Twilley) Vine

Table Queen T-AC-TQE TQEd USA (Global) Vine

Early Golden Crookneck T-CN-EAC EAC USA (Henry Field) Bush, open

Early Summer Crookneck T-CN-ESC ESCc-S USA (Ledden) Bush, open

Golden Bush Scallop T-SC-GBS GBS-1-4-2 USA (Dessert) Bush

White Bush Scallop T-SC-WBS WBS-3-Sa USA (Mandeville) Bush

Saffron T-SN-SFF SFF-S,Sa USA (Burpee) Bush, open

Straightneck Early Yellow T-SN-SNE SNE USA (American) Bush, open

Bicolor Pear T-GO-BPR BPRa-S USA (Harris) Vine

Crown of Thorns T-GO-CRT CRTa USA (Henry Field) Vine

Shenot’s Crown of Thorns T-GO-SHC SHCc USA (Jung) Vine

Spoon T-GO-SPN SPNd USA (Harris) Vine

PI 285213 T-GT-213 PI 285213-Sa,Sb USA (North Central P.I.S.) Vine

Wild Texas T-GT-WTX WTX-10-Sf,Si USA (Texas A&M Univ.) Vine

Wild Mexico 2 F-GF-WM2 WMX2-Sa Tamaulipas, Mexico

(Texas A&M U.)

Vine

Flat U-GU-FLA FLAa Canada (Stokes)b Vine

Flat Striped U-GU-FLS FLS Canada (Stokes) Vine

Miniature Ball U-GU-MNB MNB-5 Canada (Stokes) Vine

Green Warted Q-GQ-

GWA

GWA USA (Agway) Vine

Small Flat Warted Q-GQ-SFW SFW-S,Sa USA (Park)c Vine

Little Gem P-GP-LGM LGM-S,Sc South Africa (MayFord) Vine

Orange P-GP-ORA ORA-22 USA (Harris) Vine

Orange Ball P-GP-ORB ORB-3 Canada (Stokes) Vine

Orange Warted P-GP-OWA OWA-1,4 Canada (Stokes) Vine

Rolet P-GP-ROL ROL-Sa,Sb South Africa (Unknown) Vine

Genovese P-CO-GNV GNVc-S,Sa France (Vilmorin) Bush, open

Lungo Bianco di Sicilia P-CO-LBS LBS-S Italy (S.A.I.S.) Bush

Long Cocozelle P-CO-LCO LCO-1,3,7 USA (Ledden) Bush, open

Romanesco P-CO-ROM ROM-3 Italy (S.A.I.S.) Bush

Striato d’Italia P-CO-STI STIa-1,25 Italy (S.A.I.S.) Bush, open

Valery P-CO-VAA VAA Italy (La Semiorto Sementi) Vine

Verte non-coureuse d’Italie P-CO-VNI VNIc France (Delbard) Bush, open

PI 165018 P-CO-018 PI165018-Sa Turkey (North Central P.I.S.) Bush

PI 379307 P-CO-307 PI379307-5-3,7 Kosovo (North Central P.I.S.) Bush

PI 261610 P-CO-610 PI261610-S,Sa Spain (North Central P.I.S.) Vine

PI 175710 P-CO-710 PI175710-Sa,Sb Turkey (North Central P.I.S.) Bush

Citrouille de Touraine P-PU-CIR CIR-Sa France (Ducrettet) Vine

Connecticut Field P-PU-CTF CTFa USA (Ledden) Vine

Jack O’Lantern P-PU-JOL JOL-1-21 USA (Excel) Vine

Mogango sul Mineiro P-PU-MOG MOG-S,Sb Brazil (Feltrin)d Vine

Nonkadi P-PU-NOK NOK-23,S Uzbekistan (I.V.S.Q.) Vine

Porqueira P-PU-PRQ PRQ-S,Sa,Sb Portugal (Unknown) Vine

Genet Resour Crop Evol

123

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Table 1 continued

Accession name Accession

abbreviation

Accession pedigreea Origin (seed source) Plant growth

habit

Small Sugar P-PU-SSU SSUd-1 USA (Ledden) Vine

Tender and True P-PU-TAT TAT-S UK (Sutton’s) Bush

Uzbekistan Local Pumpkin P-PU-ULP ULP-S Uzbekistan (N.I.P.B.) Vine

Winter Luxury P-PU-WLU WLUa USA (Jung) Vine

Yugoslavia 7 P-PU-YU7 YU7 Former Yugoslavia (Unknown) Vine

PI 212000 P-PU-000 PI212000-S,Sa Iran (North Central P.I.S.) Vine

PI 311102 P-PU-102 PI311102 Guatemala (North Central P.I.S.) Vine

PI 442298 P-PU-298 PI442298-S Mexico (North Central P.I.S.) Vine

PI 442309 P-PU-309 PI442309-S Mexico (North Central P.I.S.) Vine

PI 442313 P-PU-313 PI442313-S Mexico (North Central P.I.S.) Vine

PI 169473 P-PU-473 PI169473 Turkey (North Central P.I.S.) Vine

PI 171628 P-PU-628 PI171628-SR,Sa,Sb Turkey (North Central P.I.S.) Vine

PI 458750 Guicoy Grande Verde P-PU-750 PI458750 Guatemala (North Central P.I.S.) Vine

All Green Bush P-VM-AGB AGB-7 UK (Asmer) Bush

Bolognese P-VM-BOG BOG-1,8 Italy (C.C.S.A.) Bush, open

Sihi Lavan P-VM-SLA SLA-26-17-1-1-29 Israel (Aratan) Bush

Table Dainty P-VM-TDA TDA-5 UK (Sutton’s) Vine

Verte Petite d’Alger P-VM-VPA VPAa-1,3 France (Abondance) Bush, open

Vegetable Spaghetti P-VM-VSP VSPe-S Japan (Sakata) Vine

Yakor P-VM-YAK YAK-2 Russia (U.N.E.E.S.S.O.K.) Bush

PI 525179 Alexandria P-VM-179 PI525179-S,Sa,Sb Egypt (North Central P.I.S.) Bush

M2546 P-VM-546 M2546-Sd,e Israel (P.I.S.) Vine

PI 181764 P-VM-764 PI181764-3 Lebanon (North Central P.I.S.) Bush

Fordhook Zucchini P-ZU-FZU FZUc-2-S USA (Burpee) Bush, open

True French P-ZU-TRF TRF-4-2-1-24-18-2-20-20-3-

16

UK (Thompson and Morgan) Bush, open

Zucchini Select P-ZU-ZSL ZSL-2-6-S Canada (Stokes) Bush, open

Tripartite designation is for subspecies, cultivar-group, and abbreviated accession name, respectively. For subspecies: T = texana,

F = fraterna, P = pepo. Other designations used for some of the accessions with regard to their subspecies affiliations are

U = uncertain and Q = questionable. For cultivar-group: AC = Acorn, CN = Crookneck, SC = Scallop, SN = Straightneck,

GO = Ovifera Gourd, GT = Texana (wild) Gourd, GF = Fraterna (wild) Gourd, GU = Uncertain Gourd, GQ = Questionable

Gourd, GP = Pepo Gourd, CO = Cocozelle, PU = Pumpkin, VM = Vegetable Marrow, ZU = Zucchinia Accession pedigrees begin with three-letter abbreviations or introduction numbers. Those not followed by hyphens indicate original

seed stocks. The number of hyphens following the three-letter abbreviations or introduction numbers indicates the number of

generations of self- or sib-pollination. A number following a hyphen indicates self-pollination whilst a capital letter ‘‘S’’ following a

hyphen, with or without a lower-case letter, indicates a sib-pollination. For example, for ‘Sihi Lavan’ (P-VM-SLA), SLA-26-17-1-1-

29 indicates that the seed stock used was derived from 5 generations of self-pollination. For ‘Fordhook Zucchini’ (P-ZU-FZU), FZU-

2-S indicates that the seed stock used was derived by one generation of self-pollination followed by one generation of sib-pollination.

For ‘Wild Texas’ (T-GT-WTX), WTX-10-Sf,Si indicates that the seed stock used was derived by one generation of self-pollination

followed by one generation of sib-pollination, but the second (sib-pollinated) generation actually consists of seeds derived from two

separate sib-pollinations that were bulkedb ‘Flat’ (U-GU-FLA) is a true-breeding derivative taken from ‘Small Warted Blend’c ‘Small Flat Warted’ (Q-GQ-SFW) is a true-breeding derivative from ‘Ovifera’d ‘Mogango sul Mineiro’ (P-PU-MOG), although sold by a Brazilian seed company, has the strong ribbing characteristic of

Guatemalan and Mexican pumpkins

Genet Resour Crop Evol

123

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(Promega, Madison, WI), and 7 ng template DNA.

Amplifications were carried out for 40 cycles in a

PTC-200 thermocycler (MJ Research, Watertown,

MA) for 60 s to denature the DNA at 92 �C, 70 s for

primer annealing at 35, 40, 45, 48, 50, 55, or 60 �C (as

determined for each primer in Table 2, based on

primer melting temperature-Tm), and 120 s for primer

extension at 72 �C. The fragments were analyzed

using a CEQ 8800 DNA Genetic Analysis System

(Beckman Coulter, Fullerton, CA) which has high

accuracy, distinguishing between DNA fragments

ranging in size from 75 to 450 base pairs and differing

in size by one base pair (Levi et al. 2010). For

visualization of DNA fragments on the CEQ 8800, the

forward primers were labeled with one of three

WellRED dye labels (D2, D3, or D4; Proligo, Boulder,

CO) as described for SRAP markers (Levi et al. 2006).

Marker data collection and analysis

The HFO–TAG fragments (Table 2) were scored as

‘‘1’’ (present) or ‘‘0’’ (absent) using the built-in

fragment analysis software provided with the Beck-

man Coulter (Fullerton, CA) CEQ-8800 Genetic

Analysis System. The resulting binary matrix was

uploaded to the PAST program version 2.17 (Hammer

et al. 2001). Dissimilarities among accessions were

calculated using Euclidean distances, which are pre-

ferred for comparisons involving dominant markers in

populations within a single diploid species (Kosman

and Leonard 2005). From the similarity matrix, a

dendrogram was constructed using the unweighted

pair-group average (UPGMA) clustering method. The

robustness of the phylogenetic tree was evaluated by

bootstrap analysis with 5,000 replicates using the

bootstrap function of PAST. Principal component

analyses (PCA) were carried out using the multivariate

data analysis of PAST. Distributions of the accessions

were depicted in two-dimensional scatter plots using

the first and second principal coordinates.

Results

Dissimilarity values

Dissimilarity values among the 74 accessions (68

Cucurbita pepo, 4 Citrullus, and 2 Lagenaria) ranged

from 0.00 for two accessions of Cucurbita pepo

pumpkins, PI 442309 (P-PU-309) and PI 442313 (P-

PU-313), to over 16.00 between many of the C. pepo

and two of the Citrullus lanatus accessions (Table 3).

The average dissimilarities among accessions

within taxa (P, T, F, Q, U, C, L) were always smaller

than those between taxa, except for one. This one

exception was within Cucurbita pepo, in which the

two Q accessions were slightly more distant from each

Fig. 1 Mature fruits of 33 accessions of Cucurbita pepo subsp.

pepo (P) and five other accessions of questionable (Q) or

uncertain (U) subspecific status. Left to right, with their

dimensions (cm, polar diameter 9 equatorial diameter): top

row, four pumpkins, P-PU-NOK (18 9 24), P-PU-CTF

(20 9 21), P-PU-PRQ (29 9 28), P-PU-000 (17 9 27); second

row, five pumpkins, P-PU-TAT (14 9 17), P-PU-WLU

(13 9 16), P-PU-MOG (16 9 13), P-PU-628 (15 9 14),

P-PU-473 (22 9 18); third row, eight vegetable marrows,

P-VM-VSP (22 9 13), P-VM-BOG (20 9 11), P-VM-SLA

(20 9 8), P-VM-546 (32 9 11), P-VM-AGB (28 9 12),

P-VM-TDA (23 9 11), P-VM-179 (32 9 14), P-VM-764

(25 9 9); fourth row, eight cocozelles and three zucchinis,

P-CO-610 (52 9 12), P-CO-VAA (42 9 10), P-CO-ROM

(40 9 11); P-CO-018 (47 9 10), P-CO-710 (39 9 11), LCO

(42 9 7), P-CO-STI (49 9 9), P-CO-LBS (41 9 8), P-ZU-TRF

(33 9 8), P-ZU-FZU (41 9 10), P-ZU-ZSL (40 9 10); bottom

row, five pepo gourds, two questionable gourds, and three

uncertain gourds, P-GP-ROL (6 9 6), P-GP-LGM (6 9 6),

P-GP-ORA (6 9 6), P-GP-ORB (7 9 6), P-GP-OWA (6 9 9),

Q-GQ-GSW (5 9 7); Q-GQ-SFW (5 9 7), U-GU-FLS

(5 9 7), U-GU-FLA (4 9 5), U-GU-MNB (4 9 4)

Genet Resour Crop Evol

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Table 2 The 22 high frequency oligonucleotide–targeting

active gene (HFO–TAG) primer sequences, their melting

(Tm) and annealing (Ta) temperatures (�C) used in PCR

reactions with genomic DNA of each of the accessions, and the

number of polymorphic fragments (NF) and their sizes

Primer Sequence Tm Ta NF Fragment sizes

HFO-13 50TCCGCCGC 38.4 45 5 122, 164, 202, 238, 242

HFO-14 50GCGGCGGA 38.4 45 17 126, 142, 149, 155, 157, 170, 172, 182, 217, 218, 226, 238, 245, 250,

261, 317, 365

HFO-19 50TCGCCGCC 38.4 45 25 82, 84, 96, 121, 125, 130, 134, 135, 137, 139, 140, 145, 148, 149, 175,

176, 178, 181, 232, 239, 262, 268, 280, 282, 284

HFO-20 50GGCGGCGA 38.4 45 10 87, 105, 110, 132, 180, 208, 211, 214, 282, 293

HFO-23 50ACGGCGGC 39.1 45 16 101, 109, 112, 167, 176, 182, 195, 212, 226, 233, 234, 272, 275, 282,

286, 305

HFO-31 50 CGCCGCCA 39.1 45 12 110, 113, 120, 121, 132, 138, 148, 198, 214, 252, 324, 325

HFO-32 50 TGGCGGCG 39.1 45 18 157, 159, 175, 176, 181, 190, 193, 207, 214, 215, 217, 220, 221, 255,

276, 293, 295, 328

HFO-37 50 CGGCGCCG 40.9 45 15 123, 134, 135, 174, 181, 183, 185, 190, 192, 198, 201, 209, 238, 243,

246

HFO-44 50 CGCCGGCG 40.9 45 12 114, 123, 127, 164, 165, 173, 211, 238, 248, 251, 325, 329

HFO-49 50 GCGGCGGT 39.1 45 32 129, 134, 153, 170, 176, 210, 211, 214, 217, 219, 221, 223, 226, 250,

253, 256, 261, 280, 283, 288, 291, 294, 302, 303, 305, 324, 326,

335, 345, 374, 376, 378

HFO-50 50 ACCGCCGC 39.1 45 15 113, 147, 157, 178, 202, 213, 237, 242, 275, 281, 294, 302, 327, 332,

361

HFO-63 50 GCCGGCGA 38.4 45 17 104, 106, 110, 113, 130, 144, 148, 178, 187, 198, 219, 221, 239, 242,

247, 260, 300

HFO-64 50 TCGCCGGC 38.4 45 20 107, 108, 148, 170, 175, 176, 178, 179, 180, 184, 190, 191, 194, 201,

204, 206, 238,

HFO-65 50 TCCGGCGG 36.7 45 13 115, 140, 192, 194, 196, 212, 229, 231, 246, 251, 254, 296, 313

HFO-66 50 CCGCCGGA 36.7 45 41 87, 97, 114, 128, 134, 148, 151, 154, 162, 167, 171, 193, 222, 224,

226, 228, 233, 235, 236, 237, 238, 241, 244, 245, 247, 249, 265,

269, 277, 285, 289, 309, 317, 327, 329, 347, 350, 362, 367, 370, 382

HFO-67 50 GCCGCTGC 36.8 45 37 82, 100, 109, 111, 123, 150, 168, 175, 179, 182, 188, 195, 197, 207,

210, 215, 220, 238, 246, 275, 284, 295, 304, 309, 315, 317, 325,

332, 337, 352, 355, 357, 373, 383, 399, 403

HFO-68 50 GCAGCGGC 36.8 45 24 122, 138, 147, 157, 161, 164, 174, 179, 183, 185, 211, 218, 222, 225,

229, 237, 242, 246, 256, 272, 275, 282, 298, 318

HFO-71 50 CCACCGCCG 42.4 45 22 93, 94, 104, 123, 149, 156, 178, 181, 184, 192, 200, 208, 223, 245,

262, 275, 286, 292, 304, 315, 327, 330

HFO-72 50 CGGCGGTGG 42.4 45 17 86, 122, 125, 136, 146, 213, 220, 222, 231, 257, 261, 282, 300, 308,

334, 344, 349

HFO-75 50 CCGCCGGC 41.9 45 42 75, 121, 133, 135, 147, 156, 167, 176, 177, 181, 187, 188, 189, 193,

194, 196, 201, 202, 203, 204, 208, 209, 210, 212, 213, 223, 271,

273, 281, 282, 318, 324, 326, 332, 336, 342, 344, 350, 382, 388

HFO-76 50 GCCGGCGG 41.9 45 19 98, 109, 111, 117, 118, 191, 195, 210, 218, 224, 228, 237, 246, 253,

274, 316, 361, 390, 393

HFO-77 50 CCTCCGCCG 41.2 45 110 60, 61, 63, 64, 65, 66, 67, 68, 69, 70, 71, 76, 79, 81, 84, 85, 87, 89, 93,

94, 96, 97, 98, 99, 101, 102, 104, 106, 107, 112, 113, 114, 116, 120,

121, 123, 125, 127, 130, 132, 133, 136, 137, 139, 143, 145, 149,

152, 156, 157, 160, 163, 164, 166, 170, 172, 173, 174, 175, 176,

178, 183, 185, 187, 188, 190, 191, 192, 195, 196, 198, 199, 204,

207, 208, 215, 216, 217, 218, 223, 229, 231, 234, 235, 241, 250,

257, 262, 270, 283, 285, 290, 291, 293, 303, 307, 309, 314, 334,

341, 348, 377, 378, 386

Genet Resour Crop Evol

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other than they were, on average, from the one F

accession (Table 3). Citrullus and Lagenaria were

closer to each other than either was to Cucurbita pepo.

The accessions within all of the subspecific Cucurbita

pepo taxa had greater average dissimilarities

(9.23–10.30) than did those of Lagenaria (5.10) and

Citrullus (8.62).

The average dissimilarities among accessions

within subsp. texana (T) and within subsp. pepo

(P) were lower than the average among accessions of

different subspecies (Table 3). The accession of

Cucurbita pepo subsp. fraterna gourd (F) was closer

to subsp. texana (T) than to subspecies pepo (P). The

‘‘uncertain’’ gourds, U, were closer on average to the

two major subspecies (T and P) than these two

subspecies were to each other.

With few exceptions, the Cucurbita pepo groups

had smaller within-group than between-group dissim-

ilarities (Table 4). One exception was the Vegetable

Marrow Group (P-VM), which had a within-group

dissimilarity equal to its dissimilarity to the Zucchini

Group (P-ZU). The Zucchini Group, though, had one

of the lowest within-group dissimilarities and by far

the lowest for the groups within subspecies pepo.

Another exception is that the two wild subsp. texana

gourds, T-GT, were more distant to each other than

they were, on average, from the two Crookneck Group

accessions, T-CN. The Crookneck Group, though, had

a low within-group dissimilarity. The other exceptions

involve the Questionable Gourds (Q-GQ), its two

representatives being more distant from each other

than they were, on average, from seven other groups.

Thus, these two gourds, while having some phenotypic

similarities, do not in fact form a cohesive group.

Except for this case, the groupings for the rest of

C. pepo are thus validated.

The within-group dissimilarities of the edible-

fruited groups of subsp. texana, T-AC, T-CN, T-SC,

Table 3 Average dissimilarities among subspecific groupings

of Cucurbita pepo, and Lagenaria siceraria and Citrullus

T F U Q P L C

T 9.44 10.11 10.42 10.40 11.42 14.04 16.00

F 10.11 – 10.87 10.26 10.64 13.75 15.69

U 10.42 10.87 9.23 10.37 10.65 14.25 16.20

Q 10.40 10.26 10.37 10.30 10.34 14.16 15.89

P 11.42 10.64 10.65 10.34 9.28 14.46 16.44

L 14.04 13.75 14.25 14.16 14.46 5.10 13.78

C 16.00 15.69 16.20 15.89 16.44 13.78 8.62

T = Cucurbita pepo subsp. texana, F = Cucurbita pepo

subsp. fraterna, U = Cucurbita pepo Uncertain Gourds,

Q = Cucurbita pepo Questionable Gourds, P = Cucurbita

pepo subsp. pepo, L = Lagenaria siceraria, C = Citrullus

lanatus and Citrullus colocynthis

Table 4 Average dissimilarities among and within groups of Cucurbita pepo

T-AC T-CN T-SC T-SN T-GO T-GT F-GF U-GU Q-GQ P-GP P-CO P-PU P-VM P-ZU

T-AC 7.07 10.88 11.35 9.18 10.54 9.87 9.80 11.90 10.93 11.10 11.95 11.95 11.69 11.39

T-CN 10.88 6.63 8.66 10.02 9.39 8.77 10.86 9.49 10.47 11.25 11.35 11.66 11.51 12.19

T-SC 11.35 8.66 6.56 10.02 9.37 9.56 11.55 9.66 10.31 11.37 11.19 11.70 11.39 11.89

T-SN 9.18 10.02 10.02 6.00 9.15 9.48 9.00 10.77 10.09 10.46 11.39 11.34 11.15 10.92

T-GO 10.54 9.39 9.37 9.15 8.74 9.05 10.03 10.40 10.21 11.02 11.34 11.41 11.28 11.11

T-GT 9.87 8.77 9.56 9.48 9.05 8.94 9.50 10.34 10.59 11.17 11.35 11.43 11.26 11.08

F-GF 9.80 10.86 11.55 9.00 10.03 9.50 - 10.87 10.26 10.32 10.85 10.79 10.62 9.54

U-GU 11.90 9.49 9.66 10.77 10.40 10.34 10.87 9.23 10.37 10.51 10.44 10.79 10.70 10.69

Q-GQ 10.93 10.47 10.31 10.09 10.21 10.59 10.26 10.37 10.30 10.19 10.28 10.55 10.25 9.80

P-GP 11.10 11.25 11.37 10.46 11.02 11.17 10.32 10.51 10.19 9.34 9.84 9.75 9.70 9.48

P-CO 11.95 11.35 11.19 11.39 11.34 11.35 10.85 10.44 10.28 9.84 8.83 9.27 8.96 8.94

P-PU 11.95 11.66 11.70 11.34 11.41 11.43 10.79 10.79 10.55 9.75 9.27 9.24 9.30 9.42

P-VM 11.69 11.51 11.39 11.15 11.28 11.26 10.62 10.70 10.25 9.70 8.96 9.30 8.92 8.92

P-ZU 11.39 12.19 11.89 10.92 11.11 11.08 9.54 10.69 9.80 9.48 8.94 9.42 8.92 6.21

T-AC = Acorns, T-CN = Crooknecks, T-SC = Scallops, T-SN = Straightnecks, T-GO = Ovifera Gourds, T-GT = Texana

Gourds, F-GF = Fraterna Gourd, U-GU = Uncertain Gourds, Q-GQ = Questionable Gourds, P-GP = Pepo Gourds,

P-CO = Cocozelles, P-PU = Pumpkins, P-VM = Vegetable Marrows, P-ZU = Zucchinis

Genet Resour Crop Evol

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and T-SN, were generally lower than those of the

edible-fruited groups of subsp. pepo, T-CO, T-PU,

T-VM, and T-ZU (Table 4). Of the edible-fruited

groups, the Pumpkins (P-PU) had clearly the highest

within-group dissimilarity, 9.24. The gourds of these

two subspecies, T-GO and T-GT for subsp. texana and

P-GP for subsp. pepo, had the highest within-group

values of their respective subspecies.

The 19 accessions of the Pumpkin Group, P-PU, are

derived from four distinct geographical regions,

southern North America (Mexico and Guatemala)

(P-PS), northern North America (USA and Canada)

(P-PN), Europe (P-PE), and Asia (P-PA). Average

dissimilarity among the accessions from southern

North America is lower than the average dissimilar-

ities between them and the pumpkins from the other

sub-groups as well as from all other groups of subsp.

pepo (Table 5). However, the pumpkins from northern

North America have a higher average dissimilarity

among themselves than they have, on average, with

the pumpkins of Europe (P-PE) and Asia (P-PA) and

therefore they do not form a cohesive sub-group. On

the other hand, the pumpkins from Asia (P-PA) and the

pumpkins from Europe (P-PE) have higher dissimi-

larities among themselves than between them; there-

fore these two geographically differentiated sub-

groups are genetically one and the same. The coco-

zelles (P-CO), vegetable marrows (P-VM), and zuc-

chinis (P-ZU) are more closely related to the European

and Asian pumpkins (P-PE, P-PA) than they are to the

North American pumpkins (P-PS, P-PN).

When the pumpkins derived from northern North

America are separated into two sub-groups on the

basis of fruit size and fruit-flesh quality, P-PP (large

ornamental fruits, low quality flesh, accessions P-PU-

CTF and P-PU-JOL) and P-PQ (small fruits, high

quality flesh, accessions P-PU-SSU and P-PU-WLU),

they show lower within-sub-group dissimilarity than

all their respective between-group dissimilarities

(Table 6). The dissimilarity between the high quality

‘Winter Luxury’ (P-PU-WLU) and ‘Small Sugar’ (P-

PU-SSU) is, however, still the highest among the sub-

groups of edible-fruited cultivars. The sub-group

having the lowest average dissimilarities, with both

the large and small pumpkins from the United States,

is the Old World pumpkins (P-PO) and the most

distant, on average, are the groups of gourds (U-GU

and Q-GQ) and the sub-group of pumpkins from

southern North America (P-PS).

The pumpkins of southern North America, P-PS,

are most dissimilar to the gourds and to the pumpkins

of the northern North America, P-PP and P-PQ. Their

lowest average dissimilarities are with the vegetable

marrows, P-VM (9.01), zucchinis, (P-ZU (9.17), and

cocozelles, P-CO (9.17). These values are distinctly

lower, in most cases, than those of the P-PP and P-PQ

pumpkins with these three squash groups. Interest-

ingly, the Old World pumpkins, P-PO, which form a

distinct sub-group with a average dissimilarity of only

8.61, have generally low dissimilarity to all of the

edible-fruited groups and sub-groups of subsp. pepo.

Their lowest dissimilarities are with the zucchinis,

Table 5 Average dissimilarities among groups and pumpkin geographical sub-groups within Cucurbita pepo subsp. pepo

U-GU Q-GQ P-GP P-CO P-PS P-PN P-PE P-PA P-VM P-ZU

U-GU 9.23 10.37 10.51 10.44 10.83 10.69 10.90 10.67 10.70 10.69

Q-GQ 10.37 10.30 10.19 10.28 11.03 10.45 10.33 10.39 10.25 9.80

P-GP 10.51 10.19 9.34 9.84 10.25 9.71 9.45 9.52 9.70 9.48

P-CO 10.44 10.28 9.84 8.83 9.28 9.74 8.89 9.17 8.96 8.94

P-PS 10.83 11.03 10.25 9.28 7.56 10.29 9.70 9.53 9.57 10.47

P-PN 10.69 10.45 9.71 9.74 10.29 9.60 9.10 9.22 9.80 9.52

P-PE 10.90 10.33 9.45 8.89 9.70 9.10 8.78 8.56 8.93 8.37

P-PA 10.67 10.39 9.52 9.17 9.53 9.22 8.56 8.85 9.01 9.17

P-VM 10.70 10.25 9.70 8.96 9.57 9.80 8.93 9.01 8.92 8.92

P-ZU 10.69 9.80 9.48 8.94 10.47 9.52 8.37 9.17 8.92 6.21

U-GU = Uncertain Gourds, Q-GQ = Questionable Gourds, P-GP = Pepo Gourds, P-CO = Cocozelles, P-PS = Pumpkins from

southern North America (Mexico and Guatemala), P-PN = Pumpkins from northern North America (USA and Canada),

P-PE = Pumpkins from Europe, P-PA = Pumpkins from Asia, P-VM = Vegetable Marrows, P-ZU = Zucchinis

Genet Resour Crop Evol

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P-ZU (8.81), vegetable marrows, P-VM (8.97), coco-

zelles, P-CO (9.05), and ornamental pumpkins from

the northern North America, P-PP (9.09). Moreover,

the sub-group of Old World pumpkins (P-PO) has

distinctly lower average dissimilarities to all of the

sub-groups of North American pumpkins than do the

pumpkins of northern (P-PP, P-PQ) and southern

North America (P-PS) to each other.

Of the North American pumpkins, the sub-group

that is overall nearest the cocozelles, vegetable

marrows, and zucchinis is the southern sub-group (P-

PS) followed by the small-fruited, quality sub-group

(P-PQ). However, this order of closeness is reversed in

relation to the Old World pumpkins (P-PO), the large

pumpkins of northern North America (P-PP) being

closest to them.

Dendrogram

As expected, the most distinct clusters in the dendro-

gram are those formed by each of the three respective

genera (Fig. 2). The four accessions of Citrullus, the

two accessions of Lagenaria, and the 68 accessions of

Cucurbita form clusters supported by 100 % boot-

strappings. Of the four accessions of Citrullus, the sole

accession of colocynth, C-CC-015, is outlying to the

three accessions of watermelons and, of the latter, the

citron watermelon, C-LA-341, is outlying to the two

dessert watermelons, C-LA-BDI and C-LA-CHG.

The Cucurbita pepo portion of the dendrogram

consists of two major clusters (Fig. 2). One of the

clusters includes all of the subspecies texana, T,

accessions, the wild subsp. fraterna gourd, F, acces-

sion and, in outlying positions, three small cultivated

gourd accessions designated as questionable, Q, and

uncertain, U. The other cluster includes all of the

subspecies pepo, P, accessions, a Q gourd, and a U

gourd in an outlying position.

Within the cluster comprised mostly of subsp.

texana, T, accessions, those of the same edible-fruited

cultivar-group associate closely with one another,

supported by bootstrappings of at least 90 % (Fig. 2).

The two Acorn Group accessions (T-AC-TQE and

T-AC-RAC) closely associate, as do the two acces-

sions of the Straightneck Group (T-SN-SFF and T-SN-

SNE), Scallop Group (T-SC-GBS and T-SC-WBS),

and Crookneck Group (T-CN-EGC and T-CN-ESC).

The non-edible, gourd, cultivars are more widely

distributed within the T cluster and, except for two

cultivars of the Ovifera Gourd Group (T-GO-BPR and

T-GO-CRT) do not associate as closely.

Two sub-clusters appear within the subsp. texana,

T, cluster. The Acorn Group (T-AC) accessions sub-

cluster with those of the Straightneck Group (T-SN)

whilst the Scallop Group (T-SC) accessions sub-

cluster with those of the Crookneck Group (T-CN)

(Fig. 2). The wild subsp. texana gourds (T-GT-WTX

and T-GT-213) are split between the sub-clusters, as

Table 6 Average dissimilarities among groups and pumpkin geographical and phenotypic subgroups within Cucurbita pepo subsp.

pepo

U-GU Q-GQ P-GP P-CO P-PO P-PP P-PQ P-PS P-VM P-ZU

U-GU 9.23 10.37 10.51 10.44 10.77 10.48 10.91 10.67 10.70 10.69

Q-GQ 10.37 10.30 10.19 10.28 10.36 10.95 9.95 10.39 10.25 9.80

P-GP 10.51 10.19 9.34 9.84 9.49 9.83 9.60 9.52 9.70 9.48

P-CO 10.44 10.28 9.84 8.83 9.05 9.75 9.72 9.17 8.96 8.94

P-PO 10.77 10.36 9.49 9.05 8.61 9.09 9.24 9.60 8.97 8.81

P-PP 10.48 10.95 9.83 9.75 9.09 8.43 9.99 10.07 10.08 9.92

P-PQ 10.91 9.95 9.60 9.72 9.24 9.99 9.11 10.04 9.53 9.13

P-PS 10.67 10.39 9.52 9.17 9.60 10.07 10.04 8.85 9.01 9.17

P-VM 10.70 10.25 9.70 8.96 8.97 10.08 9.53 9.01 8.92 8.92

P-ZU 10.69 9.80 9.48 8.94 8.81 9.92 9.13 9.17 8.92 6.21

U-GU = Uncertain Gourds, Q-GQ = Questionable Gourds, P-GP = Pepo Gourds, P-CO = Cocozelles, P-PO = Pumpkins from the

Old World (Europe and Asia), P-PP = Pumpkins from northern North America (USA and Canada), decorative, large, and of low

quality, P-PQ = Pumpkins from northern North America (USA and Canada), small and of high quality, P-PS = Pumpkins from

southern North America (Mexico and Guatemala), P-VM = Vegetable Marrows, P-ZU = Zucchinis

Genet Resour Crop Evol

123

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are the four accessions of the Ovifera Gourd Group (T-

GO). The subsp. fraterna gourd (F-GF-WM2) associ-

ates with one of the wild subsp. texana gourds (T-GT-

213).

Within the cluster comprised mostly of subsp. pepo,

P, accessions, cultivars of the same group tended to

associate with one another (Fig. 2). Generally, though,

bootstrap support was lower among the subsp. pepo

Fig. 2 UPGMA

dendrogram of 68

accessions of Cucurbita

pepo, four accessions of

Citrullus, and two

accessions of Lagenaria,

with bootstrap values

of [50 indicated

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123

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accessions than among the subsp. texana accessions.

The three accessions of the Zucchini Group (P-ZU)

sub-clustered with one another, supported by fairly

high bootstrapping. Four strongly ribbed pumpkins

from Guatemala and Mexico, P-PU-298, -309, -313,

and -MOG, also sub-clustered with one another. One

sub-cluster containing 10 accessions consisted only of

cultivars of the Cocozelle Group (P-CO) and the

Vegetable Marrow Group (P-VM), and this sub-

cluster associated with the one consisting of four

strongly ribbed pumpkins. Another sub-cluster con-

taining 10 accessions consisted almost entirely of

pumpkins and gourds.

Principal component analyses

The scatter plot based on principal component analysis

for Cucurbita pepo (Fig. 3) shows the 14 accessions of

subsp. texana, T, as distant from the 48 accessions of

subsp. pepo, P. The six accessions labeled F (subsp.

fraterna), Q (questionable), and U (uncertain) are in

intermediate positions between the T and P accessions.

Of the 14 accessions of subsp. texana, T, eight

belong to its four edible-fruited cultivar-groups, two

from each. The two accessions of each of these four

pairs are close to each other (Fig. 3). The accessions of

gourds, both cultivated (T-GO) and wild (T-GT),

though, are more widely dispersed. The cultivars of

the Acorn and Straightneck Groups are closer to one

another than they are to cultivars of the Crookneck and

Scallop Groups, which are also close to one another.

The scatter plot based on principal component

analysis of the 48 C. pepo subsp. pepo, P, accessions

(Fig. 4), shows all three zucchinis (P-ZU), FZU, TRF,

and ZSL, close to one another in the upper left

quadrant. The five gourds (P-GP), though, are widely

scattered. The ten vegetable marrows (P-VM) are

divided into two congregations, six of the accessions

-5

-4

-3

-2

-1

0

1

2

3

4

5

-8 -6 -4 -2 0 2 4

Com

pone

nt 2

(13.

0%)

Component 1 (16.4%)

Q-GQ-SFW

T-SN-SFFT-AC-TQET-SN-SNE T-AC-RAC

F-GF-WM2T-GT-213

T-GO-BPR

T-GO-CRT

U-GU-MNB

T-GT-WTXT-SC-GBS

T-GO-SPNT-CN-EGC

T-SC-WBS

T-CN-ESC

T-GO-SHC

U-GU-FLA

Q-GQ-GWAU-GU-FLS

Q-GQ-SFW

T-SN-SFFT-AC-TQET-SN-SNE T-AC-RAC

F-GF-WM2T-GT-213

T-GO-BPR

T-GO-CRT

U-GU-MNB

T-GT-WTXT-SC-GBS

T-GO-SPNT-CN-EGC

T-SC-WBS

T-CN-ESC

T-GO-SHC

U-GU-FLA

Q-GQ-GWAU-GU-FLS

Q-GQ-SFW

T-SN-SFFT-AC-TQET-SN-SNE T-AC-RAC

F-GF-WM2T-GT-213

T-GO-BPR

T-GO-CRT

U-GU-MNB

T-GT-WTXT-SC-GBS

T-GO-SPNT-CN-EGC

T-SC-WBS

T-CN-ESC

T-GO-SHC

U-GU-FLA

Q-GQ-GWAU-GU-FLS

Q-GQ-SFW

T-SN-SFFT-AC-TQET-SN-SNE T-AC-RAC

F-GF-WM2T-GT-213

T-GO-BPR

T-GO-CRT

U-GU-MNB

T-GT-WTXT-SC-GBS

T-GO-SPNT-CN-EGC

T-SC-WBS

T-CN-ESC

T-GO-SHC

U-GU-FLA

Q-GQ-GWAU-GU-FLS

Q-GQ-SFW

T-SN-SFFT-AC-TQET-SN-SNE T-AC-RAC

F-GF-WM2T-GT-213

T-GO-BPR

T-GO-CRT

U-GU-MNB

T-GT-WTXT-SC-GBS

T-GO-SPNT-CN-EGC

T-SC-WBS

T-CN-ESC

T-GO-SHC

U-GU-FLA

Q-GQ-GWAU-GU-FLS

Q-GQ-SFW

T-SN-SFFT-AC-TQET-SN-SNE T-AC-RAC

F-GF-WM2T-GT-213

T-GO-BPR

T-GO-CRT

U-GU-MNB

T-GT-WTXT-SC-GBS

T-GO-SPNT-CN-EGC

T-SC-WBS

T-CN-ESC

T-GO-SHC

U-GU-FLA

Q-GQ-GWAU-GU-FLS

Q-GQ-SFW

T-SN-SFFT-AC-TQET-SN-SNE T-AC-RAC

F-GF-WM2T-GT-213

T-GO-BPR

T-GO-CRT

U-GU-MNB

T-GT-WTXT-SC-GBS

T-GO-SPNT-CN-EGC

T-SC-WBS

T-CN-ESC

T-GO-SHC

U-GU-FLA

Q-GQ-GWAU-GU-FLS

Q-GQ-SFW

T-SN-SFFT-AC-TQET-SN-SNE T-AC-RAC

F-GF-WM2T-GT-213

T-GO-BPR

T-GO-CRT

U-GU-MNB

T-GT-WTXT-SC-GBS

T-GO-SPNT-CN-EGC

T-SC-WBS

T-CN-ESC

T-GO-SHC

U-GU-FLA

Q-GQ-GWAU-GU-FLS

Q-GQ-SFW

T-SN-SFFT-AC-TQET-SN-SNE T-AC-RAC

F-GF-WM2T-GT-213

T-GO-BPR

T-GO-CRT

U-GU-MNB

T-GT-WTXT-SC-GBS

T-GO-SPNT-CN-EGC

T-SC-WBS

T-CN-ESC

T-GO-SHC

U-GU-FLA

Q-GQ-GWAU-GU-FLS

Q-GQ-SFW

T-SN-SFFT-AC-TQET-SN-SNE T-AC-RAC

F-GF-WM2T-GT-213

T-GO-BPR

T-GO-CRT

U-GU-MNB

T-GT-WTXT-SC-GBS

T-GO-SPNT-CN-EGC

T-SC-WBS

T-CN-ESC

T-GO-SHC

U-GU-FLA

Q-GQ-GWAU-GU-FLS

Q-GQ-SFW

T-SN-SFFT-AC-TQET-SN-SNE T-AC-RAC

F-GF-WM2T-GT-213

T-GO-BPR

T-GO-CRT

U-GU-MNB

T-GT-WTXT-SC-GBS

T-GO-SPNT-CN-EGC

T-SC-WBS

T-CN-ESC

T-GO-SHC

U-GU-FLA

Q-GQ-GWAU-GU-FLS

Q-GQ-SFW

T-SN-SFFT-AC-TQET-SN-SNE T-AC-RAC

F-GF-WM2T-GT-213

T-GO-BPR

T-GO-CRT

U-GU-MNB

T-GT-WTXT-SC-GBS

T-GO-SPNT-CN-EGC

T-SC-WBS

T-CN-ESC

T-GO-SHC

U-GU-FLA

Q-GQ-GWAU-GU-FLS

Q-GQ-SFW

T-SN-SFFT-AC-TQET-SN-SNE T-AC-RAC

F-GF-WM2T-GT-213

T-GO-BPR

T-GO-CRT

U-GU-MNB

T-GT-WTXT-SC-GBS

T-GO-SPNT-CN-EGC

T-SC-WBS

T-CN-ESC

T-GO-SHC

U-GU-FLA

Q-GQ-GWAU-GU-FLS

Q-GQ-SFW

T-SN-SFFT-AC-TQET-SN-SNE T-AC-RAC

F-GF-WM2T-GT-213

T-GO-BPR

T-GO-CRT

U-GU-MNB

T-GT-WTXT-SC-GBS

T-GO-SPNT-CN-EGC

T-SC-WBS

T-CN-ESC

T-GO-SHC

U-GU-FLA

Q-GQ-GWAU-GU-FLS

Q-GQ-SFW

T-SN-SFFT-AC-TQET-SN-SNE T-AC-RAC

F-GF-WM2T-GT-213

T-GO-BPR

T-GO-CRT

U-GU-MNB

T-GT-WTXT-SC-GBS

T-GO-SPNT-CN-EGC

T-SC-WBS

T-CN-ESC

T-GO-SHC

U-GU-FLA

Q-GQ-GWAU-GU-FLS

Q-GQ-SFW

T-SN-SFFT-AC-TQET-SN-SNE T-AC-RAC

F-GF-WM2T-GT-213

T-GO-BPR

T-GO-CRT

U-GU-MNB

T-GT-WTXT-SC-GBS

T-GO-SPNT-CN-EGC

T-SC-WBS

T-CN-ESC

T-GO-SHC

U-GU-FLA

Q-GQ-GWAU-GU-FLS

Color code of P accessions:Blue = P-CO (Cocozelle)Light Green = P-VM (Vegetable Marrow)Dark Green = P-ZU (Zucchini)Red = P-GP (Gourd)Purple = P-PO (Old World Pumpkin)Dark Orange = P-PQ (North American Qlty. Pumpkin)Light Orange = P-PP (North American Orn. Pumpkin)Yellow = P-PS (Southern North American Pumpkin)

Fig. 3 Scatter plot generated by principal component analysis of 68 accessions of Cucurbita pepo

Genet Resour Crop Evol

123

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toward the left and four toward the extreme right. Six

of the 11 cocozelles (P-CO) associate with the latter,

so the upper right quadrant contains exclusively ten

accessions derived from these two cultivar-groups.

The remaining five cocozelles are more widely

scattered. Of the pumpkins, the two small, quality

(P-PQ) pumpkins are toward the extreme left, distant

from the large, ornamental (P-PP) pumpkins and from

pumpkins of southern North America (P-PS), most of

which are at the extreme lower right. In contrast, the

Old World pumpkins (P-PO) gravitate more toward

the center, nearest the ornamental pumpkins (P-PP),

and appear to be the closest sub-group to the

cocozelles (P-CO) and vegetable marrows (P-VM).

Discussion

HFO–TAG markers have been used to obtain an

improved definition of relationships in Citrullus spp.

germplasm and have contributed to mapping the

watermelon genome (Levi et al. 2010, 2011, 2013).

In PCR experiments examining the ability of primers

to amplify fragments from a watermelon cDNA

library, the HFO–TAGs primers produced consider-

ably more fragments (an average of 6.44 fragments per

primer) compared with ISSR and RAPD primers (an

average of 3.59 and 2.49 fragments per primer,

respectively). Indeed, HFO–TAG markers had far

greater capability than RAPD and ISSR markers for

revealing polymorphisms, readily distinguishing

among various dessert watermelon cultivars and

indicating relationships that are in close agreement

with known pedigrees (Levi et al. 2010). For Cucur-

bita pepo, the HFO–TAG markers provide results that

are consistent with those obtained using other nuclear

DNA markers (Lebeda et al. 2007; Formisano et al.

2012; Gong et al. 2012), but in addition reveal

relationships concerning the evolution of its econom-

ically more important lineage, C. pepo subsp. pepo.

-5

-4

-3

-2

-1

0

1

2

3

4

5

-4 -3 -2 -1 0 1 2 3 4 5

TRF

FZU

ZSL

TAT018

CIRVAA

ORB

WLU

SSU

BOGNOK

LGM

546 179764

YAK 473ROM

750VSP YU7

307

610

ROL

LBS

309, 313

CTF ULP

OWA102

JOL 628

ORA 000

PRQMOG

298

710

STI

LCO VPAAGB

TDA

GNV

VNI

SLA

Component 1 (15.6%)

Com

pone

nt 2

(8.9

%)

Color code:Blue = CO (Cocozelle)Light green = VM (Vegetable Marrow)Dark green = ZU (Zucchini)Red = GP (Gourd)Purple = PO (Old World Pumpkin)Dark Orange = PQ (North America Quality Pumpkin)Light Orange = PP (North America Ornamental Pumpkin)Yellow = PS (Southern North America Pumpkin)

Fig. 4 Scatter plot generated by principal component analysis of 48 accessions of Cucurbita pepo subsp. pepo

Genet Resour Crop Evol

123

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The HFO–TAG markers present Cucurbita pepo as

more distant from Citrullus and Lagenaria (average

dissimilarities 16.04 and 14.13, respectively) than

either of those two genera are from each other (average

dissimilarity 13.78; Table 3). Both Citrullus and

Lagenaria are likely natives of Africa (Robinson and

Decker-Walters 1997) and of the tribe Benincaseae

whilst Cucurbita is one of 11 genera in the tribe

Cucurbitae, the distribution of which is almost entirely

in the Americas (Schaefer and Renner 2011).

As expected, the HFO–TAGs clearly show the

dichotomy within Cucurbita pepo, between subsp.

texana and subsp. pepo (Table 3; Fig. 2). The HFO–

TAGs also show that the smallest-fruited gourds,

labelled Q (Questionable) and U (uncertain), are

generally intermediate between the two major culti-

vated subspecies (Tables 3 and 4; Fig. 3), as has been

observed using AFLPs and SSRs (Paris et al. 2003;

Gong et al. 2012).

The HFO–TAGs are, in general, consistent with the

horticultural classification into cultivar-groups based

mainly on fruit shape (Paris 1986). Nonetheless, the

results differ in some ways from those obtained with

other nuclear markers. Presently, a dichotomy is

observed within C. pepo subsp. texana above the level

of cultivar-group, with the acorn squash, straightneck

squash, and some gourds separated from the crook-

neck squash, scallop squash, and other gourds

(Table 4; Figs. 2 and 3). The four edible-fruited

cultivar-groups of subsp. pepo, Cocozelle, Pumpkin,

Vegetable Marrow, and Zucchini, are distinguishable

from one another by having lower within-group

dissimilarity values than their respective between-

group dissimilarity values (Table 4). They are thus

more clearly separated than observed with various

other nuclear DNA markers (Katzir et al. 2000; Ferriol

et al. 2003; Paris et al. 2003; Formisano et al. 2012;

Gong et al. 2012), but nonetheless the first three are not

completely separated in the dendrogram (Fig. 2) and

scatter plots (Figs. 3 and 4). However, analysis of the

present results with HFO–TAG markers reveals for the

first time why the cocozelles, pumpkins, and vegetable

marrows are not completely separated from one

another and can explain how the long-fruited groups,

the cocozelles and vegetable marrows, evolved.

Of the cultivar-groups of Cucurbita pepo subsp.

pepo, the Pumpkin Group contains the highest within-

group dissimilarity (Table 4). This is to be expected as

the Pumpkin Group is the oldest of the edible-fruited

cultivar-groups (Paris 2000), having originated

approximately 10,000 years ago (Smith 1997; Tepp-

ner 2004). Pumpkins having the same geographical

origin and set of phenotypic characteristics would be

expected to be more closely related to one another than

to pumpkins of different geographical origins and

appearance. Indeed, the pumpkin accessions of south-

ern North America, which bear fruits of modest size

(usually \5 kg) and are often striped and strongly

ribbed, usually with thick lignified rinds, differ greatly

in appearance from the pumpkins of northern North

America, which are grooved with non-lignified rinds,

and orange without stripes (Paris 2000; Teppner

2000). The pumpkins derived from different regions

of the North American continent are genetically quite

distant from one another (Table 5). The pumpkin

cultigens of northern North America, though, are split

among themselves into two distinct sub-groups

(Table 6) which can be distinguished phenotypically

by differing fruit size and quality. One of these sub-

groups, consisting of ‘Small Sugar’ and ‘Winter

Luxury’, has smaller fruits with high-quality flesh

and are usually consumed. The other, consisting of

‘Connecticut Field’ and ‘Jack-O-Lantern’, has larger

fruits (usually [5 kg) with low-quality flesh that are

usually used as ornaments for Hallowe’en. The three

pumpkin sub-groups from North America became

differentiated from one another on that continent

before the arrival of Europeans, probably long before

1492. All three are abundantly depicted in botanical

herbals of the Renaissance (Paris 2000; Teppner 2000)

and two of them appear in festoons produced in Rome

between 1515 and 1518 (Janick and Paris 2006). All of

the pumpkins are ultimately derived from southern

North America (Decker-Walters 1990) but it is not

known where and when the two more northerly sub-

groups of pumpkins were founded. Perhaps each was

derived through gradual diffusion northward, with

selection occurring in the United States for orange

external color and larger size or improved culinary

value. All three of the North American sub-groups are

closer to the sub-group of Old World (Euro-

pean ? Asian) pumpkins than they are to one another

(Table 6).

The Old World pumpkins encompass wide pheno-

typic variation (Paris 2000; Ferriol and Pico 2008),

ranging in size from medium, approximately 2 kg, to

quite large, 20 kg or more (Fig. 1). Most have thin

lignified rinds and are not grooved. Many are ribbed,

Genet Resour Crop Evol

123

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but usually not as prominently as the pumpkins of

southern North America. Many have dark green

stripes on an orange, yellow, or light green back-

ground. They flower early enough so as to be able to

set and ripen their fruits in short-season areas of

Europe and Asia. Phenotypically, therefore, the Old

World pumpkins possess a combination of traits which

is intermediate to that possessed by pumpkins from the

southern part versus the northern part of North

America. The genetic intermediacy of the Old World

pumpkins (Table 6) is consistent with their having

originated from spontaneous crossing among pump-

kins of the different North American subgroups. The

intercrossing among pumpkin subgroups had to have

occurred after they were brought to Europe and grown

in close proximity. For example, Leonhart Fuchs, the

German botanist, had a garden in which he grew exotic

plants. He produced a large collection of plant

illustrations in approximately 1562, referred to as the

Vienna Codex (Meyer et al. 1999), among which are

images of Cucurbita pepo. Some of the C. pepo

images are clearly of inter-group hybrids (Paris 2000).

Indeed, southern and northern North American sub-

groups of pumpkins were grown in near proximity to

one another in the environs of Rome already within

25 years of the first voyage of Columbus to the

Americas (Janick and Paris 2006).

The accessions of the long-fruited cultivar-groups

of subsp. pepo, Cocozelle, Vegetable Marrow, and

Zucchini, are each more closely related to the Old

World subgroup of pumpkins than they are to any of

the North American pumpkin subgroups (Table 6).

Some cocozelle and vegetable marrow accessions also

show affinity with pumpkins from southern North

America (Fig. 2). Variant plants of southern North

American landrace pumpkins can produce elongate

fruits, but there is no evidence for the existence of

uniformly long-fruited cultivars of C. pepo subsp.

pepo in North America in the first centuries after the

European contact with that continent (Zhiteneva 1930;

Hernandez Bermejo and Leon 1994; Paris 2000;

Teppner 2000). Therefore, the development of the

long-fruited cultivars of subsp. pepo seems to have

occurred in the Old World over the past 500 years.

Iconographic records indicate that incipient cocozelles

and vegetable marrows were present in Europe within

a century of the arrival of C. pepo pumpkins from

North America (Paris 2000; Paris and Janick 2005).

Gene exchange between pumpkins from northern

North America and pumpkins from southern North

America not only led to the development of the

European and Asian pumpkins, but also to the

development of three entirely new cultivar-groups

having elongate fruits, the cocozelles, vegetable

marrows and, eventually, the zucchinis.

Selection for deviation from ancestral round fruit-

edness was not a one-time occurrence in the develop-

ment of Cucurbita pepo cultivars. In C. pepo subsp.

texana, several cultivars of the flat scallop squash are

illustrated in mid-sixteenth century herbals and the

elongate crookneck squash is also an ancient group

(Paris 2000; Teppner 2000). The de novo evolution of

long-fruited cultigens of C. pepo subsp. pepo, within a

century of the arrival of round-fruited cultigens of this

subspecies in Europe, suggests that this germplasm

underwent intensive selection there during the six-

teenth century. The deviation from ancestral fruit

roundness in the cultivated cucurbits is associated with

a greater proportion of the firm, fleshy mesocarp tissue

and colored exocarp at the expense of the relative

volume of the seedy endocarp (Sinnott and Durham

1929). Such developments are therefore associated

with abandoning the original uses of cucurbit fruits by

people, consumption of the mature fruit flesh or seeds,

in favor of consuming the whole young fruits (Paris

1989). Selection for elongated cucurbit fruits has a

long tradition in the Old World. Snake melons,

Cucumis melo L., and long bottle gourds, Lagenaria

siceraria, were already in widespread use as food by

Latin-, Greek-, and Hebrew-speaking cultures of the

Mediterranean Basin during the time of the Roman

Empire (Janick et al. 2007). Indeed, the snake melon

was the most important cucurbit crop in Mediterra-

nean lands through medieval times (Paris 2012). So it

is easily understood how, when a cucurbit newcomer

arrived in this region, Cucurbita pepo subsp. pepo, it

was directionally selected for elongate fruits. This

selection occurred most intensively in Italy but also in

Asia Minor (Gong et al. 2012) and Spain (Formisano

et al. 2012). In each of these places, the founder

populations of pumpkins from which elongate fruits

were selected may well have been different, the result

being the fuzzy demarcation between the cocozelles,

vegetable marrows, and pumpkins seen today (Figs. 2

and 4).

Of the subsp. pepo gourds, all of which are small in

size and round in shape, two cultivars from South

Africa, ‘Rolet’ and ‘Little Gem’, show affinity to each

Genet Resour Crop Evol

123

Page 16: Genetic relationships in Cucurbita pepo (pumpkin, squash, gourd) as viewed with high frequency oligonucleotide–targeting active gene (HFO–TAG) markers

other (Fig. 2). ‘Orange Ball’ is closest to the Ques-

tionable Gourd ‘Small Flat Warted’, one of the

smallest gourds and near the genetic center of the

species (Gong et al. 2012). Although ‘Orange’ and

‘Orange Warted’ are closest to several pumpkins

(Figs. 2 and 4), neither they nor any of the P-GP,

Q-GQ, or U-GU gourds seems to have made a

significant contribution to the radiation of the edible-

fruited groups and sub-groups of subsp. pepo, as they

are distant (dissimilarity [ 9.40) to all of them

(Tables 4, 5, and 6).

Conclusions

Polymorphisms of HFO–TAG markers, which are

derived mostly from coding regions of the genome,

have resulted in an assessment of genetic diversity

within Cucurbita pepo that closely corresponds with

the morphological variation exhibited by this species.

The four edible-fruited cultivar-groups of C. pepo

subsp. texana are clearly distinguished, Acorn, Crook-

neck, Scallop, and Straightneck. The four edible-

fruited cultivar-groups of C. pepo subsp. pepo, Coco-

zelle, Pumpkin, Vegetable Marrow, and Zucchini, are

more clearly differentiated by the HFO–TAGs than

heretofore seen with other DNA markers, but are

nonetheless not as distinct as those of C. pepo subsp.

texana. A dichotomy was observed within C. pepo

subsp. texana above the level of cultivar-group, with

the acorn squash, straightneck squash, and some

gourds separated from the crookneck squash, scallop

squash, and other gourds. In C. pepo subsp. pepo, three

populations of North American pumpkins are differ-

entiated, one southerly and two northerly, one of the

latter having small, high-quality fruits and the other

having large, ornamental fruits. Apparently, these

three pumpkin sub-groups had been isolated from one

another on their native continent, but records indicate

that they were grown in close proximity in Europe

soon after the initial contacts of European explorers

with various parts of North America over 500 years

ago. The present results show that the cocozelles,

vegetable marrows, and zucchinis are more closely

allied with the Old World cultivars of pumpkins than

with any of the North American source populations.

Acknowledgments We thank Laura Massey of the USDA–

ARS, Charleston, for expert technical assistance.

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