Germplasm collection and
characterization in tomatillo
(Physalis philadelphica Lam.)
Todd Vision
Department of Biology
University of North Carolina at Chapel Hill,
USA
Solanaceae in North Carolina
Tomatillo
(Physalis philadelphica Lam.)
• Annual
• Self-incompatible
• Fruit enclosed by a
papery calyx
• Native to Mexico
• Diploid (n=12)
from Hernández Bermejo and León 1994
Ethnobotany of tomatillo
• Culinary uses
• Salsa verde (usually with
Capsicum)
• Infusion of calyx to make
tamale dough
• Medicinal uses
• Leaves and fruits for
headaches and
stomachaches
• Juice for sore throats
• Cooked calyx for
diabetes
Economic importance
• Cultivation mostly in
Mexico and Guatemala
• In Mexico
• Fifth most important
vegetable
• > 25,000 Ha cultivated/yr
• Elsewhere
• Equal importance in
Guatemala
• Used by growing Mexican
population in U.S.A. (esp.
California)
• Used internationally in haute
cuisine
Phylogeny of Solanaceae
Petunia, Brunfelsia
Nicotianeae (Nicotiana)
Anthocercideae
Jaboroseae
Nolanea
Lycieae
Hyoscyameae
Nicandreae
Datureae
Solaneae (Solanum)
Capsiceae (Capsicum)
Physaleae (Physalis)
Solandreae
Mandragoreae
From Olmstead et al. 1999 Solanaceae IV
M. Whitson (2002)
Two-gene phylogeny of the Physalinae
rbcL and ITS
Leucophysalis viscosa
Brachistus spp., Witheringia spp.
Tzetalia spp
P. alkekengi (Chinese lantern)
Ocryctes spp, Leucophysalis nana, L. grandiflora
P. microphysa
P. arborescens
Quincula lobata
Chamaesaracha spp.
P. crassifolia, P. acutifolia
Margaranthus
New World clade
P. philadelphica (tomatillo)
P. microcarpa, P. ignota, P. cordata, P. lagascae, P. pruinosa,
P. angulata, P. pubescens
P. nicandroides, P. peruviana (uchua), P. chenopodifolia,
P. coztomatl, P. sordida, P. hederifolia, P. glutinosa,
P. longifolia, P. greenmanii, P. caudella
U.S. perennials
P. minima, P. lanceolata, P. hetrophylla, P. virginina, P. arenic
P. pumila, P. mollis, P. viscosa, P. cinerascens, P. angus
P. walteri
Nomenclature
• Physalis philadelphica Lam. (= P. ixocarpa Brot.)
• tomate from Nahuatl ayacach tomatl meaning berry
• Local names:
•
•
•
•
•
miltomate (Oaxaca)
tomate verde (Jalisco)
tomatillo (Jalisco, Oaxaca, Puebla)
tomate de cascara (Jalisco, Puebla, Oaxaca, Chiapas)
tomate de hoja (Jalisco, Puebla)
• tomate de milpa, or miltomate, from its association
with maize fields
Domestication
• Cultivation is ancient
• Remains in the Valley of
Tehuacán in Puebla
• Gradient of cultivation still
visible
• But few modern cultivars
• Primitive features of
landraces
•
•
•
•
Self-incompatibility
Indeterminate growth
Nonsynchronous ripening
Fruit drop
• Lack of clear wild relative
Fruit size variation
Classification of varieties
• Work of Santaguillo Hernández and
Peña Lomelí (Universidad Autónoma
Chapingo)
• Mostly morphological
• Some molecular (AFLP) evidence
• Three most important varieties
• Rendidora (central and southern Mexico)
• Salamanca (Guatemala)
• Tamazula (in western Mexico)
variety
habit
fruiting
yield
fruit
fruit color
calyx color
Rendidora
crawling
early
high
medium
lemon green
green
Salamanca
erect
late
mediu
m
large
deep green
clear green
Tamazula
erect
early
mediu
m
medium
purple
green to
purple
Puebla
verde
crawling to
semierect
early
mediu
m
large
green
green with
purple veins
Manzano
crawling to
semierect
late
mediu
m
large
orange
green
Arandas
erect
early
low
small to
medium
green to
purple
green to
purple
Milpero
cultivado
crawling to
erect
late
very
low
very small
green to
purple
green purple
Milpero no
cultivatado
crawling to
erect
late
very
low
very small
green,
yellow,
purple
green to
purple
SF1
Chapingo
crawling to
semierect
very
early
very
high
medium
lemon green
green
Summary of Hudson work
• Self-incompatibility and crossability
• Model of domestication
Germplasm conservation
2002 Collecting Expedition
Sponsored by USDA Plant Exchange
Office
Maria Chacon and Todd Vision
University of North Carolina
Ofelia Vargas Ponce
Universidad de Guadalajara
Larry Robertson
USDA-ARS
Aureliano Peña Lomelí
Universidad Autónoma Chapingo
Andrew Jarvis
CIAT
Existing collections
MEXICO
seedbank
herbarium
both
Jalisco
Michoacan
Puebla
Guerrero
Oaxaca
Chiapas
Predicted priorities from
FloraMap
Sources of collection
•
•
•
•
•
•
Undisturbed vegetation (none)
Disturbed vegetation
Tomatillo fields
Other fields (maize)
Traditional markets
Farmer’s seed stock
Classification of collections
• Uncultivated
• From undisturbed or disturbed habitat, or a weed in a
field occupied by other crops (maize)
• Incipient domesticate
• Casually but intentionally cultivated in fields devoted to
other crops (maize)
• Landrace
• Cultivated for self-consumption or sale at a local
market
• Escape
• Resembling a landrace but not found in a cultivated
field
2002 collections
Jalisco
Puebla
Michoacán
Guerrero
Oaxaca
uncultivated
incipient domesticate
escape
landrace
Chiapas
2002 collections
State
Chiapas
Guerrero
Hidalgo
Jalisco
Michoacán
Oaxaca
Puebla
Uncult.
6
8
3
4
-
Incip.
1
9
-
Escape
1
3
-
Landrace Collection sites
22
6
6
12
2
1
4
12
1
3
25
12
16
8
Total
21
10
4
76
54
Comparison of Mexican
collections
State
Baja
California
Chiapas
Chihuahua
Colima
Guanajuato
Guerrero
Hidalgo
Jalisco
México
Michoacán
Morelos
Nayarit
Oaxaca
Puebla
San Luis
Potosí
Sonora
Zacatecas
Total
BANGEV
No. of
No. of
accessions
collection
sites
3
1
USDA
No. of
accessions
-
No. of
collection
sites
-
New Collections
No. of
No. of
accessions
collection
sites
-
1
1
7
7
4
74
26
21
9
9
100
125
1
1
1
6
4
3
45
10
14
6
7
1
16
1
8
4
-
5
4
-
22
13
2
13
3
39
11
-
18
11
1
13
2
37
8
-
1
2
391
1
2
119
12
9
105
90
P. angulata
P. ampla
Local preferences
•
•
•
•
Jalisco: purple fruit
Puebla: large, green
Guerrero: yellow
Chiapas: small and
purple
Field trials
• Larry Robertson at
USDA Plant Genetic
Resources Unit
(Geneva, New York)
• Tested 99 accessions
from 2002 collection
• 4 plants per accession,
one in a pollinatorexclusion cage
• Measured a suite of
agronomic and
domestication traits
Results of field trials
•
•
•
•
•
•
Domestication characters
Agronomic characters
BRIX
Selfing
Yield
Interest from organic farmers
Uses of microsatellite markers
• Genetic diversity
• Phylogeography and the origin of
domestication
• Paternity analysis and varietal
fingerprinting
• Genetic mapping
• Marker-assisted breeding
Microsatellites
Simple Sequence Repeats (SSR), Short Tandem Repeats
(STR)
Mono (A)11
AAAAAAAAAAA
Di (AT)8
ATATATATATATATAT
Tri (ATC)7
ATCATCATCATCATCATCATC
Tetra (CTAG)6 CTAGCTAGCTAGCTAGCTAGCTAG
Imperfect
Interrupted
Compound
GTGTGTGTATGTGTGT
GTGTGTGTCCCGTGTGTGT
GTGTGTGTCTCTCTCTCTCT
Microsatellites: advantages
•
•
•
•
•
•
Abundant
Codominant
Highly polymorphic
Highly repeatable
PCR-based
Can be multiplexed
for amplification or
scoring
Development of SSR markers
• Two strategies
• From enriched SSR libraries
• From tomato SSRs
Enrichment of SSRs
Digest genomic DNA & ligate adapters
Hybridize to biotin-labeled SSR probes
Capture with magnetic streptavidin beads
Clone captured fragments
Sequence inserts and design primers
Screen primers for polymorphism (in 8 genotypes)
Enriched library results
• 11 libraries generated
• 1620 positive clones isolated
• 659 clones sequenced (w/ inserts 500-1500
bp).
• 205 (31%) clones contained one or more
SSR
• SSRs represented 40 unique loci
• Primers designed for 24 loci
• Eight were easily scorable and highly
polymorphic
Enriched library results
motif
ACT
AAT
AAG/AT C
AT G/T T C
TTG
AGT
TTA
AAC
AC
AG
AT
T OT AL
clones isolatedclones with
inserts 5001500 bp
43
25
117
60
67
28
0
0
248
32
126
69
89
20
50
11
609
249
0
0
271
165
1620
659 (41%)
clones with
SSR
3
4
23
0
21
31
2
10
52
0
59
205 (31%)
SSR motif(rep eat Unique
contigs
n u mber)
9
(AC)5 -2 7
2
(AG)5 -7
17
(AAC)4 -2 5
1
(AAT 4)
2
(AT C)5 -7
1
(AAAC)4
Compound 7
Imperfect
1
T otal
40
P rimer pairs
4
2
8
1
2
1
5
1
24
Transferring tomato SSRs
• Primers developed for SSRs in tomato
expressed sequence tags (from SGN,
http://sgn.cornell.edu)
• 24/87 (27.5%) primers amplified a product in
tomatillo
• ~25% of di- and tri-nucleotides
• One out of nine among compound SSRs
• Six products were
• Approximately the expected size
• Yielded clean, bright bands
• Two were highly polymorphic
Tomatillo microsat markers
Cross-amplification of primers
Species
P. acutifolia
P. angulata
P. ampla
P. pruinosa
P. peruviana
P. alkekengi
Solanum
lycopersicum
Solanum
melongena
Capsicum
annuum
Nicotiana
tabaccum
MIC3 MIC48 MIC89
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
MIC98 MIC242
X
X
X
X
X
X
X
X
X
X
X
X
MIC269 MIC301 MIC303
X
X
X
X
X
X
MIC341
X
X
X
MIC350
X
X
X
SSR43 SSR140
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
Tests of Mendelian
segregation
• Segregation tested using ~50 F2
progeny per marker
• All 12 markers showed nuclear
segregation
• One (SSR140) showed significant
distortion
• No linkage detected among markers
• But not all marker combinations tested
Genetic diversity survey
• Is there evidence for a genetic
bottleneck within cultivated genotypes?
• Is there evidence for restricted gene
flow
• Among geographic regions?
• Between cultivated and uncultivated
genotypes?
• What is the geographic origin of
cultivated genotypes?
Survey sample
• Includes germplasm from
• USDA
• BANGEV (Mexico)
• CATIE (Costa Rica, samples from Guatemala)
• 69 genotypes
• 39 different sites
• 19 sites represented by 2 or 3 genotypes
• 6 Guatemalan states and 15 Mexican states
• Cultivation status
• 21 uncultivated and 39 cultivated (all Mexican)
• 9 unknown (all Guatemalan)
Diversity and cultivation status
Locus
MIC3
MIC48
MIC89
MIC98
MIC242
MIC269
MIC301
MIC303
MIC341
MIC350
SSR43
SSR140
0.47
Mean
Cultivated
A
He
16
0.89
7
0.65
4
0.54
2
0.03
2
0.12
6
0.70
15
0.86
4
0.56
19
0.89
9
0.65
13
0.87
9
8.8
0.6
Uncultivated
A
13
5
4
2
2
7
13
5
13
7
10
0.47
7.1
He
0.89
0.68
0.45
0.13
0.13
0.61
0.82
0.54
0.86
0.76
0.84
4
0.6
Physiographic provinces
Northern states
Trans-Mexican Volcanic Belt
Chiapas-Guatemala
Sierra Madre del Sur
© 1975 Board of Regents, The University of Texas
Population structure
N: Northern states,
T: Trans-Mexican Volcanic
Belt, S: Sierra Madre del Sur
C: Chiapas-Guatemala
0.1
Weedy
Cultivated
Analysis of Molecular
Variation
By geographic province
source
among groups
within groups
d.f.
3
132
SumSq
19.07
437.72
total
135
456.79
%Var
2.78
97.22
By cultivation status
source
among groups
within groups
total
d.f.
1
118
119
SumSq
8.14
400.04
408.18
%Var
2.5
97.5
Population structure analysis
• Bayesian statistical approach for
detecting admixed populations
(Pritchard et al. 2000)
• Assigns individuals fractionally to K
subpopulations
• Optimal model is K=2 (with prob~0.98)
• Six pairs of markers show significant
gametic disequilibrium
Diffuse domestication: a
hypothesis
• Domestication in
multiple regions?
• Extensive gene flow
with uncultivated
populations after
domestication?
Future genetic work
• EST sequencing
• Tod evelop molecular markers
• Crosses among self-compatible accessions
• To map the genetic markers
• Identify QTL for key domestication and agronomic
traits
• Association mapping of candidate genes
• Shallow population structure
• High levels of diversity and recombination
Prospects for improvement
• Traits of importance
• Determinate growth
• Self-compatibility
• Resistance to
lodging
• Fruit retention
• Loose calyx
Pests and diseases
•
•
•
•
Viruses
Powdery mildew
Coleoptera
Heliothis subflexa
caterpillars
Conclusions
• Much potential for crop improvement of
tomatillo
• Germplasm resources are now available
• Variation for some traits (self-incompatibility) but not
others (indeterminate growth)
• A panel of 12 microsatellites are now available
• Very little population structure
• Possibly diffuse domestication
• Extensive gene flow
• Excellent system for candidate gene
association mapping
Many thanks to
• María Chacon and Maria Tsompana (UNCCH)
• Assistants Casey Kolb, Letycia Argote Nuñez,
Leah Schinasi, Lindsey Swanson
•
•
•
•
•
Larry Robertson (USDA)
Aureliano Peña Lomelí (Chapingo)
Ofélia Vargas de Ponce (Guadalajara)
Andrew Jarvis (CIAT)
Karen Williams (USDA Plant Exchange
Office)
• USDA, CATIE, and BANGEV seed banks