Post on 15-Dec-2015
Germplasm collection and characterization in tomatillo (Physalis
philadelphica Lam.)
Todd VisionDepartment of Biology
University of North Carolina at Chapel Hill, USA
Solanaceae in North Carolina
Tomatillo (Physalis philadelphica
Lam.)
from Hernández Bermejo and León 1994
• Annual • Self-incompatible• Fruit enclosed by
a papery calyx• Native to Mexico• Diploid (n=12)
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 SolanaceaePetunia, Brunfelsia
Nicotianeae (Nicotiana)
Jaboroseae
Hyoscyameae
From Olmstead et al. 1999 Solanaceae IV
Anthocercideae
Nolanea
Nicandreae
Solaneae (Solanum)
Datureae
Capsiceae (Capsicum)
Physaleae (Physalis)
Mandragoreae
Solandreae
Lycieae
M. Whitson (2002) Two-gene phylogeny of the Physalinae
rbcL and ITSLeucophysalis viscosaBrachistus spp., Witheringia spp.Tzetalia spp
P. alkekengi (Chinese lantern)
Ocryctes spp, Leucophysalis nana, L. grandiflora
P. microphysa
P. arborescensQuincula lobataChamaesaracha spp.
P. crassifolia, P. acutifoliaMargaranthus
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
P. minima, P. lanceolata, P. hetrophylla, P. virginina, P. arenicola, P. pumila, P. mollis, P. viscosa, P. cinerascens, P. angustoflia, P. walteriU.S. perennials
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 medium
large deep green clear green
Tamazula erect early medium
medium purple green to purple
Puebla verde
crawling to semierect
early medium
large green green with purple veins
Manzano crawling to semierect
late medium
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 ExpeditionSponsored by USDA Plant Exchange
Office
Maria Chacon and Todd Vision University of North Carolina
Ofelia Vargas Ponce
Universidad de GuadalajaraLarry Robertson
USDA-ARSAureliano Peña Lomelí
Universidad Autónoma ChapingoAndrew Jarvis
CIAT
Existing collections
MEXICO
Jalisco
Guerrero
OaxacaChiapas
PueblaMichoacan
Jalisco
seedbank herbarium both
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
Michoacán
Guerrero
Oaxaca Chiapas
Puebla
uncultivatedincipient domesticateescapelandrace
2002 collections
State Uncult. Incip. Escape Landrace Collection sitesChiapas - - - 22 6Guerrero 6 - 1 6 12Hidalgo - - - 2 1Jalisco 8 1 3 4 12Michoacán 3 - - 1 3Oaxaca 4 9 - 25 12Puebla - - - 16 8
Total 21 10 4 76 54
Comparison of Mexican collections
BANGEV USDA New CollectionsState No. of
accessionsNo. of
collectionsites
No. ofaccessions
No. ofcollection
sites
No. ofaccessions
No. ofcollection
sitesBajaCalifornia
3 1 - - - -
Chiapas 1 1 - - 22 18Chihuahua - - 8 5 - -Colima 1 1 - - - -Guanajuato 7 6 - - - -Guerrero 7 4 - - 13 11Hidalgo 4 3 - - 2 1Jalisco 74 45 - - 13 13México 26 10 4 4 - -Michoacán 21 14 - - 3 2Morelos 9 6 - - - -Nayarit 9 7 - - - -Oaxaca 100 1 - - 39 37Puebla 125 16 - - 11 8San LuisPotosí
1 1 - - - -
Sonora 1 1 - - - -Zacatecas 2 2 - - - -Total 391 119 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 pollinator-exclusion 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
Mono (A)11 AAAAAAAAAAADi (AT)8 ATATATATATATATATTri (ATC)7 ATCATCATCATCATCATCATCTetra (CTAG)6 CTAGCTAGCTAGCTAGCTAGCTAG
Imperfect GTGTGTGTATGTGTGTInterrupted GTGTGTGTCCCGTGTGTGTCompound GTGTGTGTCTCTCTCTCTCT
Simple Sequence Repeats (SSR), Short Tandem Repeats (STR)
Microsatellites
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
Sequence inserts and design primers
Screen primers for polymorphism (in 8 genotypes)
Clone captured fragments
Capture with magnetic streptavidin beads
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 clones isolated clones with inserts 500-1500 bp
clones with SSR
ACT 43 25 3AAT 117 60 4AAG/ATC 67 28 23ATG/TTC 0 0 0TTG 248 32 21AGT 126 69 31TTA 89 20 2AAC 50 11 10AC 609 249 52AG 0 0 0AT 271 165 59TOTAL 1620 659 (41%) 205 (31%)
SSR motif(repeat
number)
Unique contigs
Primer pairs
(AC)5-27 9 4
(AG)5-7 2 2
(AAC)4-25 17 8
(AAT)4 1 1
(ATC)5-7 2 2
(AAAC)4 1 1
Compound 7 5Imperfect 1 1Total 40 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 markersTa
a Mg++ Primer Size Genotypic(°C) conc. conc. ranged distort.
(mM) (µM) (bp)F:CTAACTCATCCGCTGATTCATCR:AATAATTCTCTACCCCCTCCTF:CCTTTAGGAAAAAACAAGAACTCR:TACTACACCTCTATAGCCCGATAATCF:GTCAGCGACAGATTTAAGGTTCR:GCCAACGTAGTCATTTGTCAGTF:CCTTGTCGTGGACTTTATTATCCR:CACACCTGCCTCTGAAACGF:GTTGTAGGTTAGCATATTGGGGTAGTAR:TCTATAACAGATAGAGAGGCTGTTTCCF:ATACAACCCTGCCCAAAACCR:CTCATTCTGTGTCCAGCTTCCF:TATTTTTGTCTTGAGCCGAGGR:AGAGTGTTCATGAGAACAATATGCCF:CCATTGACTGTGAAATCGTGR:CTGTTCATTAGCAGTATCAGCTCF:GTAGAACCCTGTCTTCCATAAGTGR:GACAGAGAGAAACAAGATTCAGTAGF:CACAAGAGTTAGGTCCATTTACTTGR:CAGAAAGAGAGAGATAGAGAATTGGF:TTACTAGGTACCGTGAGATGCR:ACATTCTACCCTCCCCAGACF:AGTGAGTTATCTGGTAGTCATCCTGR:TGTAAGAAACACCACTCTATGTCTCF:CTCTTCATTAGACGTTGCTCTAR:GACGAGTCACATTATTCAGTTGF:CTCACCGTAGATACAAGTAGAATACR:AGGGTTTAAAATCCCCTACATCF:CTCCAAATTGGGCAATAACAR:TTAGGAAGTTGCATTAGGCCAF:GTTTCAGCAGCCTGTCCAATR:TGACAGCATTTGGGTTTGAG
Marker Motif No. allelesd HE b F c Allelic distort.
MIC3 (AAC)855 2.5 0.5 18 233-273 0.91 0.06 3.95 9.04
MIC5 (AAC)655 2.5 0.5 1d 118d
MIC48 (ATC)550 2.5 0.4 8 220-228 0.7 0.11 1.12 1.37
MIC89 (AAT)455 2.5 0.2 5 284-340 0.53 -0.08
MIC96 (AAAC)460 2.5 0.4 1e 210d
MIC98 (AAC)560 2.5 0.4 2 187, 189 0.06 0
MIC103 (AAC)755 2.5 0.5 1e 169d
MIC242 (ATC)760 2.5 0.4 2 104, 109 0.12 -0.08
MIC269 (AAC)455 2.5 0.2 7 230-258 0.72 0.19 2.97 14.69
MIC301 (AC)755 2.5 0.4 18 322-385 0.87 0.17 4.08 6.73
MIC303 (AAC)760 2.5 0.2 5 312-383 0.52 0.58
MIC341 (CA)9(GA)650.6 2.5 0.4 22 174-221 0.89 0.14 6.86 10.5
MIC350 (AC)860 3.5 0.6 12 251-326 0.73 0.48
MIC441 (AC)550 2.5 0.4 1e 240d
SSR43 (TAC)756.6 2.5 0.4 1e 195-216 0.87 0.43 0 0
SSR140 (CAG)755 2.5 0.07 8.56 15.22*0.2 10 120-144 0.45
Cross-amplification of primers
Species MIC3 MIC48 MIC89 MIC98 MIC242 MIC269 MIC301 MIC303 MIC341 MIC350 SSR43 SSR140P. acutifolia X X X X X X X XP. angulata X X X X X X X XP. ampla X X X X X X X X XP. pruinosa X X X X X XP. peruviana X X X X X X X XP. alkekengi X XSolanum lycopersicum
X X X
Solanum melongena
X X X X
Capsicum annuum
X X X X
Nicotiana tabaccum
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
Cultivated UncultivatedLocus A He A He
MIC3 16 0.89 13 0.89MIC48 7 0.65 5 0.68MIC89 4 0.54 4 0.45MIC98 2 0.03 2 0.13MIC242 2 0.12 2 0.13MIC269 6 0.70 7 0.61MIC301 15 0.86 13 0.82MIC303 4 0.56 5 0.54MIC341 19 0.89 13 0.86MIC350 9 0.65 7 0.76SSR43 13 0.87 10 0.84SSR140 9 0.47 4 0.47
Mean 8.8 0.6 7.1 0.6
Physiographic provinces
© 1975 Board of Regents, The University of Texas
Northern states
Trans-Mexican Volcanic Belt
Sierra Madre del Sur
Chiapas-Guatemala
Population structure
0.1
N: Northern states, T: Trans-Mexican Volcanic Belt, S: Sierra Madre del SurC: Chiapas-Guatemala
WeedyCultivated
Analysis of Molecular Variation
By geographic provincesource d.f. SumSq %Varamong groups 3 19.07 2.78within groups 132 437.72 97.22
total 135 456.79
By cultivation statussource d.f. SumSq %Varamong groups 1 8.14 2.5within groups 118 400.04 97.5total 119 408.18
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 (UNC-CH)
• 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• The many farmers who shared their seeds