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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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,
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
(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
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
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
Genet Resour Crop Evol
123
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
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