GENETIC DIVERSITY, POPULATION STRUCTURE AND HOST
RESISTANCE TO PHYTOPHTHORA FRUIT ROT IN THE SOLANACEAE
By
Rachel Pearl Naegele

A DISSERTATION
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
Plant, Breeding, Genetics and Biotechnology –Doctor of Philosophy
2013

ABSTRACT
GENETIC DIVERSITY, POPULATION STRUCTURE AND
PHYTOPHTHORA FRUIT ROT RESISTANCE IN THE SOLANACEAE
By
Rachel Pearl Naegele
Production of eggplant (Solanum melongena) and pepper (Capsicum annuum), the
third and fourth most important solanaceous crops worldwide, are limited by diseases
caused by the oomycete pathogen Phytophthora capsici Leonian. In peppers, fruit rot
resistance 3 and 5 days post inoculation (dpi) was mapped in an F6 recombinant inbred
line population between a resistant, landrace Serrano and susceptible, cultivated Jalapeño.
Isolate-specific interactions were evident and 10 quantitative trait loci were identified in
the population with low to moderate effects. Diverse collections of eggplants (99) and
peppers (160) were evaluated for genetic diversity, population structure, and fruit rot
resistance to two isolates of P. capsici. In the eggplant and pepper collections, four
genetic clusters were detected by Bayesian analysis. Resistance to one or both isolates
was found for at least one accession in both collections. In the eggplant collection,
population structure was detected when individuals were grouped by the following
predefined categories: disease resistance, country of origin, continent of origin, and fruit
shape. In the pepper collection, population structure was detected when individuals were
grouped by disease resistance, country of origin, and continent of origin. These results
provide a baseline for future work utilizing global pepper and eggplant resources, and
developing Phytophthora fruit rot resistant cultivars.

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ACKNOWLEDGEMENTS
I would like to acknowledge the many individuals who contributed to the success
of this work. Specifically, I would like to thank A. Tomlinson, S. Boyle, J. Mitchell, H.
Gutting and J. Olsen who contributed technical assistance on multiple aspects of these
projects and Drs. M. Hausbeck, A. Iezzoni, R. Buell, R. Hammerschmidt, A. Van Deynze
and L. Quesada for critical evaluation of this document and their insightful suggestions.

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TABLE OF CONTENTS
LIST OF TABLES……………………………………………………………………….v
LIST OF FIGURES…………….………………………………………………………vii
LITERATURE REVIEW……………….……………………………………………....1
CHAPTER 1: QTL MAPPING OF FRUIT ROT RESISTANCE TO THE PLANT
PATHOGEN PHYTOPHTHORA CAPSICI L. IN A RECOMBINANT INBRED
LINE CAPSICUM ANNUUM L. POPULATION.……………….…………………….9
ABSTRACT………………………………………………………………………9
INTRODUCTION…….………………………………………………………..10
MATERIALS AND METHODS……………….……………………………...12
RESULTS……….………….…………………………………………………...15
DISCUSSION…………………………………………………………………...18
CHAPTER 2: GENETIC DIVERSITY, POPULATION STRUCTURE, AND
RESISTANCE TO PHYTOPHTHORA CAPSICI OF A WORLDWIDE
COLLECTION OF EGGPLANT……………………………………………………..22
ABSTRACT………………………………………………………………….....22
INTRODUCTION……………………………….……………………………..23
MATERIALS AND METHODS……………………………..………………..26
RESULTS……………………………………………………………...………..34
DISCUSSION…………………………………………………………………...47
CHAPTER 3: EVALUATION OF A DIVERSE, WORLDWIDE COLLECTION
OF WILD, CULTIVATED AND LANDRACE PEPPERS (CAPSICUM ANNUUM)
FOR RESISTANCE TO PHYTOPHTHORA FRUIT ROT, GENETIC
DIVERSITY, AND POPULATION STRUCTURE……...…………………………..53
ABSTRACT…………………………………………………………………….53
INTRODUCTION…………………….……………………………………..…54
MATERIALS AND METHODS……………………………………………....57
RESULTS………………………………………………...……………………..68
DISCUSSION…………………………………………………….……………..83
BIBLIOGRAPHY…….………………………………………………………………...86

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LIST OF TABLES
Table 1.1 Distribution and marker number for the pepper recombinant inbred line (RIL)
genetic map…………………………………………………...……………...…………..14
Table 1.2 Fruit rot resistance QTL………………………………………..……………..15
Table 1.3 Fruit phenotype QTL………….……………………………..………………..17
Table 2.1 Eggplant germplasm used for the study of morphological and molecular
variation.…………………………………………………………………………………26
Table 2.2 Fruit shape parameters and mean disease ratings for each isolate overall and
per individual experiment.……………….………………………………………………34
Table 2.3 Solanum spp. fruit shape, width and length variation between countries of
origin.…………………………………………………………………………….………38
Table 2.4 Correlation of Solanum spp. fruit characteristics with disease
susceptibility……………….…………………………………………………….………39
Table 2.5 Polymorphic primers evaluated against 99 eggplant lines………….…..…….40
Table 2.6 Genetic differentiation (pairwise Fst) estimates of SSRs for S. melongena
grouped by continent……………………………………….…………………………….41
Table 2.7 Genetic diversity estimates for SSRs for S. melongena grouped by continent
and country of origin……………………………………………………………………..41
Table 2.8 Genetic differentiation (pairwise Fst) estimates of SSRs for S. melongena
grouped by country………………………………………….……………………...……42
Table 2.9 Genetic differentiation (pairwise Fst) estimates of SSRs for eggplant
germplasm grouped by disease resistance…………………..………………………..….42
Table 2.10 Genetic differentiation (pairwise Fst) estimates of SSRs for S. melongena
germplasm grouped by fruit shape.………………………………………………………43
Table 3.1 Pepper lines used in this study.……………………………………………….57
Table 3.2 Simple sequence repeat (SSR) markers tested against the pepper genotypes and
their respective genetic diversity and polymorphism information content (PIC) within the
population.……………………………………………………………………………….65

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Table 3.3 Pepper fruit disease susceptible at 3 days post inoculation (dpi) and 5dpi when
inoculated with isolates OP97 and 12889.………………………….……………………69
Table 3.4 Capsicum spp. Phytophthora fruit rot resistance among countries of
origin.……………………………………………………………….……………………73
Table 3.5 Capsicum spp. Phytophthora fruit rot resistance among continents of
origin……………………………………………………………………………………..74
Table 3.6 Genetic diversity of pepper genotypes among countries and continents...…...80
Table 3.7 Genetic differentiation (FST pairwise differentiation) of pepper genotypes
among countries and continents…………………………………………………...……..81
Table 3.8 Genetic differentiation (FST pairwise differentiation) among pepper disease
resistance categories 3 (shaded values) and 5 days post inoculation (dpi) when inoculated
with OP97 and 12889………………………………………………………………..…..82

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LIST OF FIGURES
Figure 2.1 Eggplant Phytophthora fruit rot disease rating scale shown on various
eggplant genotypes: 0= no visible symptoms, a rating of 1=<25% symptomatic area,
25%>2<50%, 50%>3<75%, and a rating of 4!75% symptomatic area of the fruit. (For
interpretation of the references to color in this and all other figures, the reader is referred
to the electronic version of this dissertation)…………………………….………………30
Figure 2.2 Fruit size and shape differences between eggplants. S. incanum (left) and S.
linnaeanum (right) fruit (A), S. melongena fruit (B) and S. linnaeanum and S. melongena
fruit varying in shape, size and color (C). U.S. quarter used for size reference…………37
Figure 2.3 Population structure grouped by species. Cluster 1 (purple), Cluster 2 (light
yellow), Cluster 3 (sky blue) and Cluster 4 (steel blue). ………………………………..44
Figure 2.4 Population structure grouped by continent of origin for eggplant germplasm.
Cluster 1 (purple), Cluster 2 (light yellow), Cluster 3 (sky blue) and Cluster 4 (dark blue).
A white space and black tick marks separate subgroups of individuals.………………..45
Figure 2.5 Population structure grouped by country of origin for the S. melongena
germplasm. Only countries represented by four or more individuals were included.
Cluster 1 (purple), Cluster 2 (light yellow), Cluster 3 (sky blue) and Cluster 4 (dark blue).
A white space and black tick marks separate subgroups of individuals…………………45
Figure 2.6 Population structure grouped by disease resistance to isolate 12889 (A) and
OP97 (B). Individuals were grouped into a resistant and moderately resistant category
(R/MR), a moderately susceptible category (MS), and a susceptible category (S) based on
their mean disease ratings. Cluster 1 (purple), Cluster 2 (light yellow), Cluster 3 (sky
blue) and Cluster 4 (dark blue). A white space and black tick marks separate subgroups
of individuals.….………………………………………………………………………...46
Figure 2.7 Population structure grouped by S. melongena fruit shape. Cluster 1 (purple),
Cluster 2 (light yellow), Cluster 3 (sky blue) and Cluster 4 (dark blue). A white space
and black tick marks separate subgroups of individuals…………………………………47
Figure 3.1 Population structure grouped by species. Cluster 1 (purple), Cluster 2 (light
yellow), Cluster 3 (sky blue), Cluster 4 (steel blue), Cluster 5 (dark purple)…….……..76
Figure 3.2 Population structure grouped by continent of origin for pepper germplasm.
Cluster 1 (purple), Cluster 2 (light yellow), Cluster 3 (sky blue), Cluster 4 (steel blue),
Cluster 5 (dark purple). A white space and black tick marks separate subgroups of
individuals.………………………………………………………………………………76
Figure 3.3 Population structure grouped by country of origin for the C. annuum
germplasm. Only countries represented by four or more individuals were included.
Cluster 1 (purple), Cluster 2 (light yellow), Cluster 3 (sky blue), Cluster 4 (steel blue),
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Cluster 5 (dark purple). A white space and black tick marks separate subgroups of
individuals.….…………………………………………………………………………..77
Figure 3.4 Population structure grouped by Phytophthora fruit rot resistance to isolate
OP97 at A) 3 days post inoculation and B) 5 days post inoculation. Individuals were
grouped into a resistant and moderately resistant category (R/MR), a moderately
susceptible category (MS), and a susceptible category (S) based on their mean disease
ratings. Cluster 1 (purple), Cluster 2 (light yellow), Cluster 3 (sky blue), Cluster 4 (steel
blue), Cluster 5 (dark purple). A white space and black tick marks separate subgroups of
individuals.….……………………………………………………………………………78
Figure 3.5 Population structure grouped by Phytophthora fruit rot resistance to isolate
12889 at A) 3 days post inoculation and B) 5 days post inoculation. Individuals were
grouped into a resistant and moderately resistant category (R/MR), a moderately
susceptible category (MS), and a susceptible category (S) based on their mean disease
ratings. Cluster 1 (purple), Cluster 2 (light yellow), Cluster 3 (sky blue), Cluster 4 (steel
blue), Cluster 5 (dark purple). A white space and black tick marks separate subgroups of
individuals..….………………………………………………………………………….79

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LITERATURE REVIEW
The Solanaceae is a large plant family consisting of more than 2500 species in
100 genera (81). This family has a great diversity of growth habits and fruit
characteristics, but few of these species have any economic importance. Potatoes,
tomatoes, eggplants and peppers are the major solanaceous food crops and they account
for a variety of food, medicine, spices and ornamentals worldwide. A number of
important weeds including nightshade are also included in the Solanaceae. Potatoes, the
rd

3 most important crop in the world, is the most important solanaceous crop and
generates over $3.5 billion annually in the U.S. alone (USDA ERS, 2010.) Pepper and
eggplant, the fourth and third most important solanaceous crops worldwide, generate an
estimated $802 (pepper) and $42 (eggplant) million dollars annually in the U.S (USDA
NASS). China, the leading producer of both eggplants and peppers, produces 27.7 and
15.5 million tons per year, respectively (FAO, 2011).
The Solanaceae encompasses both Old and New World species. Many cultivated
solanaceous crops are New World species, originating from the Americas. Eggplant is
one of the few cultivated species from the Old World. Centers of origin and diversity are
often important sources of genetic diversity, and individuals from these areas can be used
for crop improvement. Wild and landrace individuals can harbor many important
agronomic traits including improved yield, drought and disease resistance.
The eggplant complex in its most simplified form consists of the wild and semiwild species Solanum incanum and Solanum melongena each with four groups (A-D and
E-H, respectively) (121). Domestication of the cultivated eggplant, S. melongena is
thought to have occurred in Asia as early as 59 B.C. (52,75,118). Since that time, it has
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been transported and cultivated around the world (93,121). Studies have indicated that the
progenitors of domesticated eggplant (S. melongena) originated in Africa and were
derived from the closely related S. incanum (part of the S. melongena complex) (24) and
S. linnaeanum (121). Both S. incanum and S. linnaeanum can form partially fertile
hybrids with S. melongena making them potential sources for desirable traits (24,25,46).
Pepper, similar to many New World crops, originated in Central and South
America. Domestication of pepper is estimated to have occurred between 5000 and 6000
B.C. in the Americas where it was primarily used as a spice (86,88). Upon discovery of
the Americas, peppers were transported to Europe, and subsequently the world.
Cultivated pepper consists of the five species: Capsicum annuum and C. chinense, C.
frutescens, C. baccatum, and C. pubescens (1,51,84). Crossability varies between the
species, but it is generally accepted that all cultivated species but C. pubescens are
relatively crossable (42). In the U.S., C. annuum is the primary pepper species grown,
and includes both pungent chile-type and non-pungent bell-type peppers (42,84). Mexico
is the center of origin and diversity for C. annuum (30,53). The remaining pepper species
originated in various parts of Central and South America, and are now predominantly
produced in South America or Asia (3,30,42,87). Aji peppers, Peruvian peppers and
habanero are common names of cultivated non-C. annuum species. Species, geographical
and market separations have lead to the distinct genetic pools among and between pepper
species. Studies have shown that there is variability for agronomic traits of interest
between species, but there is also variability between market classes within species
(87,104,115).

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Understanding the genetic diversity and population structure within a species is
important for efficient utilization of germplasm resources. Historically, genetic diversity
has been the traditional method for species evaluation rather than population structure. A
number of marker types have been implemented to identify individuals with the greatest
variability using genetic, isozyme, morphological, and most recently, metabolite markers
(49,51,60,104,105,115). In addition to predicting heterosis when breeding, genetic
diversity can also be used to provide an overview of the total diversity available within a
population or collection to develop “core collections”, subsets of individuals who
together represent 80% of the variation of the collection. These collections provide useful
subsets for screening individuals for particular traits of interest.
Population structure analysis has recently been gaining popularity as a method to
detect and visualize spatial and temporal differences between genetic subpopulations
(23,33,55,95,96,107). Information on population structure can provide insight about
connections between phenotypic variation and the distribution of genetic diversity.
Marker-trait evaluations, such as association mapping, rely on large populations to detect
phenotypic correlations. Genetic relatedness can have a significant effect on these studies
if population structure is not taken into account. False trait associations are common
when populations exhibit genetic structure and mathematical models have been
developed to account for this (13,58). If population structure is known prior to studies, it
can improve utilization of the germplasm through the selection of individuals. Fruit
shape, disease resistance, and other characteristics can all be affected by population
structure. If population structure is present in materials being evaluated for association
mapping, spurious associations may be made between a particular genotype and the trait

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of interest (123). Combining phenotypic values with genetic population structure can
yield useful results for association mapping and breeding.
In peppers and eggplants, significant genetic diversity and population structure
within, and between, geographic regions exists (1,2,51,52,60,77,82,83,91). In peppers,
most studies have focused on genetic diversity and population structure within the centers
of origin ((1,3,49,67,83). Demonstrated population structure between wild, semi-wild and
domesticated C. annuum in Mexico was identified (1,83). Population structure has also
been evaluated in Italian, Tunisian and Turkish populations of peppers (2,10,60,91). Each
of these studies evaluated genetic diversity and/or population structure. No studies have
evaluated the level of diversity and population structure on a global basis or linked those
results with phenotypic traits of interest. Previous studies have shown that admixture
between pepper species is limited, consistent with reports of low crossability between
some species (51,104). Similar to peppers, many studies have looked at the genetic
diversity of eggplants within specific countries or regions, and a recent study compared
genetic differentiation and structure in three countries (6,34,52,59,70,72,75,78,90,93).
Most studies have looked at eggplant diversity within a single region (6,75,78), but many
have also looked at variability between Solanum spp. to solidify the boundaries between
cultivated eggplants and their relatives (6,34,70,90). Eggplants have a high degree of
morphological plasticity making species designations difficult without the use of
molecular markers (121).
Disease resistance is an important attribute for modern cultivars of peppers and
eggplants. One disease, Phytophthora fruit rot, is caused by the destructive oomycete
Phytophthora capsici L. This pathogen can infect multiple solanaceous species including

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eggplant, pepper, and tomato (31,40,97). It can infect roots, stems, fruit and foliage of
peppers or the roots, crown and fruit of eggplants at any point during development
(47,53,61,100,116). In peppers, this disease causes losses worldwide. Eggplant is affected
by Phytophthora root rot less frequently, and fruit symptoms are the most common in the
field (37). Disease management utilizes a combination of chemical and cultural controls
to reduce losses, but economic reductions in yield can still occur under conditions
favorable for disease (40,47).
Phytophthora capsici is a generalist pathogen and has a large host range
encompassing over 50 species including most cucurbits, beans and some brassicas in
addition to the Solanaceae. This broad host range limits the effectiveness of management
using crop rotations. The pathogen boasts a polycyclic disease cycle and can persist in the
soil as thick walled oospores for 10 years (40). In many instances, both mating types of
the pathogen have been identified in an area allowing sexual (oospore) reproduction, in
addition to asexual (sporangia and zoosporangia) reproduction (20,40). Because of the
genetic variability from sexual reproduction and random mutations, this pathogen also
has the ability to quickly develop fungicide resistance in the field and overcome host
resistance (40,47,66).
Phytophthora species, commonly called water molds, are superbly adapted to
dispersal through water and water control is essential to reduce the spread and severity of
the disease (36,40,47,102). While some Phytophthoras have evolved to utilize wind
dispersion, P. capsici relies solely on water dispersion (40,41,57). During a rain or
irrigation event, the cytoplasm of a sporangium can differentiate into 20-40 two-tailed
zoospores (40,47). These zoospores can swim to a new host, attach, germinate and

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penetrate the host tissue resulting in infection (11,64,122). In peppers, cultivar and
germplasm testing for root rot resistance has identified multiple lines with resistance to
one or more isolates, but few lines exhibit resistance to all isolates (18,62,73). The broad
host range, persistent biology and genetic diversity make P. capsici incredibly difficult to
manage.
First described by L. Leonian in 1922, P. capsici was identified on pepper fruit in
New Mexico (69). Since this time, the more common root rot disease in pepper has
upstaged fruit rot and fruit rot resistance. Most studies and breeding have emphasized the
identification and implementation of genetic control through quantitative trait loci (QTL)
for the root rot symptom of the disease in peppers. Studies have estimated the number of
QTL for root rot resistance to vary drastically from a single dominant gene with
modifiers, to 14 QTL (5,12,68,80). A major QTL for root rot resistance was identified on
chromosome 5, and molecular markers have been developed for marker-assisted selection
(68,98). Development and incorporation of resistant varieties into commercial production
systems has been slow. A few tolerant varieties have been bred, but most are susceptible
to highly virulent isolates of P. capsici (31) and utilize a common source of resistance.
Criollo de Morelos 334 (CM334), a landrace from Mexico, is a small fruited Serranotype pepper with resistant to all isolates evaluated to date (18,31,38,110). Most breeding
for Phytophthora resistance and molecular evaluation of resistance has utilized this line
(101).
Comparative studies between root rot and foliar blight in peppers suggested that
there is a single dominant gene controlling much of the foliar resistance. This gene
differed from the major gene contributing to root rot resistance (109,116). These results

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were later confirmed by Ogundiwin et al. (80) in a separate mapping population where
QTL were detected for foliar blight resistance with minimal overlap with root rot
resistance. The independent associations between the foliar blight, stem blight, and root
rot symptoms of the disease were also seen in a mass greenhouse evaluation by Candole
et al. (18).
Root rot is less prevalent in eggplants, and mechanisms controlling resistance are
less well studied than those in pepper. A study examined the resistance of two eggplant
breeding lines and a single commercial cultivar to multiple isolates of P. capsici (32).
The commercial cultivar was resistant to moderately virulent isolates, but was susceptible
to highly virulent isolates. The two breeding lines had high levels of resistance to most
isolates of P. capsici evaluated.
Fruit and root rot occur in the field, but fruit rot is more common in eggplant (37).
In the field, chemical management is expensive and provides limited protection against
Phytophthora fruit. Host resistance, an important part of a successful, sustainable
management program, is not currently available for management of Phytophthora fruit rot
and currently, no known eggplant or pepper lines or cultivars are resistant. The
inheritance of fruit rot resistance in pepper has been evaluated in a single study (106). In
1978, Saini and Sharma looked at the inheritance of fruit rot in a mapping population in
the field and found that it segregated in a 3:1 Mendelian fashion. They concluded that a
single dominant gene controlled fruit rot resistance. Other studies have looked at
morphological and physiological traits correlated with fruit rot resistance in pepper
(9,111). Only cuticle thickness and reactive oxygen species production were found to be
associated with ontogenic resistance in peppers (9). In eggplant, this pathogen has been

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of minor importance and no studies have been done to identify resistant germplasm,
QTL, or markers associated with fruit rot resistance.
Pepper and eggplant are two economically important crops with a wide range of
uses worldwide. In field production, P. capsici can cause large losses in yield on both
when conditions are suitable for disease. Current cultivated varieties are susceptible to
Phytophthora fruit rot and future breeding activities should include fruit rot resistance.
Identifying resistant materials using existing germplasm resources, and characterizing the
population structure and genetic diversity of those resources is needed for identification
and efficient implementation of Phytophthora fruit rot resistance in peppers and
eggplants.

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CHAPTER 1: QTL MAPPING OF FRUIT ROT RESISTANCE TO THE PLANT
PATHOGEN PHYTOPHTHORA CAPSICI L. IN A RECOMBINANT INBRED
LINE CAPSICUM ANNUUM L. POPULATION
ABSTRACT
Phytophthora capsici is an important pepper (Capsicum annuum L.) pathogen causing
fruit and root rot, and foliar blight in field and greenhouse production. Determining the
genetic basis for fruit rot resistance will greatly improve the efficiency of incorporating
resistance into commercial cultivars. Previously, an F6 recombinant inbred line
population was evaluated for fruit rot susceptibility and isolate-specific partial resistance
were found among lines. In this study, Phytophthora fruit rot resistance was mapped in
the same F6 population between Criollo del Morelos 334 (CM334), a landrace from
Mexico, and the cultivar ‘Early Jalapeno’ using a high-density genetic map. Isolatespecific resistance was mapped independently in 66 of the lines evaluated. Heritability of
2

the resistance for each isolate at 3 days post inoculation (dpi) and 5 dpi was high h =0.63
2

to 0.68 and h =0.74 to 0.83, respectively. Significant additive and epistatic quantitative
trait loci (QTL) were identified for resistance to P.capsici isolates OP97 and 13709 (3
and 5dpi) and 12889 (3dpi only). Mapping of fruit traits showed potential linkage with
few disease resistance QTL. The partial fruit rot resistance from CM334 suggests that this
may not be an ideal source for fruit rot resistance in pepper.

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INTRODUCTION
Phytophthora capsici Leonian, is an important pathogen of pepper, Capsicum annuum, in
the U.S and worldwide. This destructive pathogen is capable of infecting roots, stems,
fruit and foliage of peppers at any point during development (47,53,61,100,116). Disease
management utilizes a combination of chemical and cultural controls to reduce losses, but
economic reductions in yield can still occur under conditions favorable for disease (40).
The pathogen’s biology, polycyclic reproduction and the development of thick walled
oospores, supports long survival in the soil, rapid development of fungicide resistance
and suppression of host resistance (40,47,66). Screening of pepper germplasm for root rot
resistance has identified multiple lines with resistance to one or more isolates, but few
lines exhibit resistance to all isolates ((18,62,73). Phytophthora capsici’s broad host
range, persistent survival structures and isolate diversity make P. capsici incredibly
difficult to manage.
Most studies and breeding have emphasized the identification and implementation
of genetic control through the use of quantitative trait loci (QTL) for the root rot
symptom of the disease. Studies have estimated the number of QTL for root rot resistance
to vary drastically from a single dominant gene with modifiers, to 14 QTL (5,68,80). A
major QTL for root rot resistance was identified on chromosome 5, and molecular
markers have been developed for marker-assisted selection (68,98). Development and
incorporation of resistant varieties into commercial production systems has been slow. A
few tolerant varieties have been bred, but most are susceptible to highly virulent isolates
of P. capsici (31). Criollo de Morelos 334 (CM334), a landrace from Mexico, is resistant
to root rot for all isolates evaluated to date (17,31,32,38,110). Most breeding for

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Phytophthora resistance and molecular evaluation of resistance has utilized this line
(101).
Breeding for the other symptoms of the disease (fruit rot and foliar blight) has
been limited. Comparative studies between root rot and foliar blight suggest that there is
a single dominant gene controlling much of the foliar resistance. This gene was different
from the major gene contributing to root rot resistance (109,116). This was later
confirmed by Ogundiwin et al (80) in a separate mapping population where QTL were
detected for foliar blight resistance with minimal overlap with root rot resistance. The
independent associations between the foliar blight, stem blight, and root rot symptoms of
the disease were also seen in a mass greenhouse evaluation by Candole et al. (17).
Resistance to fruit rot is even less studied. Commercial cultivars are susceptible to
Phytophthora fruit rot and no QTL for fruit rot resistance have been identified.
The inheritance of fruit rot resistance has been evaluated in a single study (106).
In 1977, Saini and Sharma looked at the inheritance of fruit rot in a mapping population
and found that it segregated in a 3:1 Mendelian fashion. They concluded that a single
dominant gene controlled fruit rot resistance. Previous work by Naegele et al, in a
recombinant inbred line (RIL) pepper mapping population with a different source of
resistance, demonstrated partial and isolate-specific resistance to P. capsici (Naegele and
Hausbeck (in review)).
Maintaining desirable fruit traits such as color, firmness, and gloss is important
when breeding resistance into a cultivated background from landraces and wild relatives.
Often, wild relatives are small fruited, pungent and have many characteristics not suitable
for commercial production. Landraces, though more similar to their commercial

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counterparts, often contain a number of undesirable traits. Numerous studies have looked
at correlations between fruit characteristics and Phytophthora fruit rot. Pungency,
pericarp thickness and fruit firmness were shown to not be associated with fruit rot
(9,111). Reactive oxygen species (ROS) production had a negative correlation with
disease susceptibility, and higher length to width ratio for fruit shape had a positive
correlation with fruit rot (9). Linkage of fruit rot resistance with fruit-related traits would
make incorporation of the resistance into a commercial background more difficult.
The objectives of this study were to map general and isolate-specific resistance in
CM334 to P. capsici-induced fruit rot at 3 and 5 days post inoculation in an F6
recombinant inbred line population and to identify fruit-phenotypic QTL to test for colocalization with disease resistance.

MATERIALS AND METHODS
An F2 derived F6 Early Jalapeno x Criollo de Morelos (CM334) recombinant
inbred line population consisting of 66 individuals previously screened for fruit rot
resistance ((15,94, Naegele and Hausbeck (in review)). Three isolates of P. capsici,
12889 (A1, I, pepper), OP97 (A1, S, cucumber) and 13709 (A2, IS, bean), from the
collection of Dr. Mary Hausbeck (Michigan State University) were used for inoculations.
Isolates were characterized by mating type (A1 or A2), sensitivity to mefenoxam
(I=insensitive, S=sensitive and IS=intermediately sensitive) and host of origin. In brief,
three detached immature green peppers per isolate from each line were surface
disinfested in a 10% bleach solution for 5 min and rinsed in distilled water. Fruit were
placed into a humidity chamber and inoculated with agar plugs (6 mm in diameter) from
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actively growing P. capsici colonies placed topside down on the pepper fruit and covered
with sterile, micro-centrifuge caps. Control peppers were inoculated with a V8 agar plug
and covered with a sterile, micro-centrifuge cap. Peppers were kept in the humidity
chambers under constant light at room temperature (25 ˚C). Peppers were evaluated at
early (3dpi) and late (5dpi) responses based preliminary results of the earliest symptoms
on commercial peppers and longest time to cover the fruit (data not shown). Three and
five days post inoculation, lesion width and diameter was measured for each individual
fruit using a hand caliper. The experiment was repeated two times for a total of 9 peppers
per line per isolate.

Fruit evaluations for fruit length, width and shape were previously performed as
described by Naegele and Hausbeck (in review). In brief, 20 peppers from each line were
evaluated for fruit length (cm), width (cm), shape (maximum length/width in cm), and
color (light green, green, dark green, purple). In addition twenty peppers from each line
were evaluated for gloss (low, medium, high), firmness, (1 to 3), and pericarp thickness
(in cm). Fruit length, width, shape and pericarp thickness were measured using a hand
caliper. Gloss and firmness were determined as described by Chaim et al (19).

Data were analyzed using the PROC MIXED function of the SAS v9.3 software
using the LSmeans statement (SAS Institute Cary, NC). Significant (P=0.05)
interactions between line and isolate were separated using the SLICE option. Line means
(LSmeans output) for isolate-specific lesion area 3 and 5 dpi were used for QTL
mapping. Line means for fruit gloss, firmness and pericarp thickness were calculated in

!$&!

SAS and used for QTL mapping. Narrow-sense trait heritability was estimated using the
progeny mean basis method (29,118).

QTL analysis was performed using R/QTL function in the statistical software R
(15,16,99) using the genetic map previously built by Hill et al (50). The map was
reconstructed in R/qtl. Prior to QTL mapping, the linkage map was evaluated for
recombination frequency, marker order, segregation distortion and switched genotypes.
Problematic markers were removed from the map. The final map contained 3,814
markers with 222 to 450 markers per chromosome with an estimated coverage of 1267
cM (Table 1.1).
Table 1.1. Distribution and marker number for the pepper recombinant inbred line
(RIL) genetic map
Chromosome P1 P2 P3 P4 P5 P6 P7 P8 P9 P10 P11 P12
# of markers 450 407 415 244 222 267 140 261 513 305 363 227
cM distance 140 101 124 100 78.5 122 116 86 90 107 113 89.9
Estimated coverage: 1267.2
Total number of markers per chromosome from P1 to P12 (# of markers), the
Centimorgan (cM) distance per chromosome (cM distance) and the estimated coverage of
the genetic map in cM (Estimated coverage).
Missing genotypes were identified at each marker and the genotype was estimated using
the imputation method implemented in R/qtl. QTL for each trait were identified using
interval mapping by the multiple imputation method. Significance of QTL was
determined using 1000 replicates of the permutation test. After a single significant QTL
was identified, additional additive and epistatic QTL were added using composite interval
mapping. Multiple QTL, interactions and effects of individual QTL were confirmed and

!$'!

estimated using a general linear model implemented in R/QTL where y equals the sum of
the individual QTL and their interactions (y~Q1+Q2+Q1*Q2).
RESULTS
QTL for resistance to Phytophthora fruit rot were detected for isolates 12889,
13709 and OP97 at both 3 and 5 days post inoculation. Significant LOD thresholds at
P=0.05 were 3.11 to 3.36 for disease responses. QTL for fruit characteristics had
significant LOD thresholds at P=0.05 for gloss, firmness, fruit shape and pericarp
thickness at 3.26, 3.32, 2.97 and 3.3, respectively.

At 3dpi, 34 lines were resistant or partially resistant to P. capsici (data not
shown). Heritability of disease resistance to individual isolates was 0.68, 0.63 and 0.63
for isolates 12889, 13709 and OP97, respectively. All QTL were significant at P=0.01
unless specified otherwise. Isolate-specific QTL with varying effects were detected for
each of the P. capsici isolates tested. For isolate OP97, two QTL were detected. One
QTL, located on chromosome (chr) 6 at 56 cM, explained 14.8% of the variation seen
and another on chr 5 at 21 cM, explained 12% of the trait variation. The resistant allele
on chr 6 was from the susceptible parent (‘Early Jalapeno’) (Table 1.2).
Table 1.2. Fruit rot resistance QTL
QTL with additive effects
Trait

Chr.

Pos.

12889

2

13

3dpi

6

58.1

Marker name
CAPS_CONTIG.
10457
CAPS_CONTIG.
3200

13709

5

0

CAPS_CONTIG.
10639
!$(!

Source

Sig

Est.
2
h

0.167

CM

**

0.68

6.66

0.381

EJ

***

3.36

0.164

CM

***

LOD
Value

R

3.33

2

0.63

Table 1.2 (cont’d)
CAPS_CONTIG.
10555

3dpi

6

69.3

13709

3

124

5dpi

5

71.2

OP97

5

21.1

3dpi

6

56

CAPS_CONTIG.
11660
CAPS_CONTIG.
1455

13
29.8

CAPS_CONTIG.
9283
KS25046E04

OP97
5dpi

4
5

KS24039C09
CAPS_CONTIG.
11780

5.51

0.292

EJ

***

4.4

0.207

CM

***

7.49

0.330

CM

***

5.21

0.273

CM

***

3.26

0.158

EJ

***

9.53
4.05

0.175
0.500

CM
CM

***
***

0.83

Source

Sig

0.74

0.63

QTL with epistatic effects
Trait
12889
3dpi

Chr.

Pos.

P2*P6

13*58.1

Marker name
CAPS_CONTIG.10457 *
CAPS_CONTIG.3200

13709
5dpi

P3*P5

124*71.2

KS24039C09 *
CAPS_CONTIG.11780

LOD
Value

R2

2.76

0.136

CM*EJ ***

1.24

0.070

CM*C
M

**

OP97
CAPS_CONTIG.9283 *
CM*C
5dpi
P4*P5
13*29.8
KS25046E04
3.39
0.138
M
***
Additive and epistatic effect QTL for general response resistance 3 and 5 days post
inoculation (3dpi and 5dpi, respectively) and isolate specific resistance for isolates
12889, 13709 and OP97 3 and 5dpi. Chromosome (chr) and genetic map position (Pos) of
the marker in cM. LOD value of the QTL, the percent of the variation explained by the
2
QTL (R ) and the donor parent of the positive effect allele (Source). Significance of the
QTL were determined by the general linear model implemented in R/QTL at P=0.05 (*),
P=0.01 (**), and P=0.001 (***). Estimated narrow sense heritability of the trait
2
calculated using variance components (Est. h ).
Resistance to 13709 was also correlated with two QTL. One QTL, on chr 6 at
69.3 cM, explained almost 30% of the variation and came from the susceptible parent
while the other at the tip of chr 5 (0 cM) was from the resistant parent and explained
17%. Isolate 12889 also had contributions for resistance from both parents at chr 6 (58.1

!$)!

cM) and chr 2 (13 cM) explaining 38 and 16.4% of the variation, respectively. An
interaction between the two QTL contributed another 13%.
At 5dpi most lines evaluated were susceptible to P. capsici. Isolate specific
responses were detected for each of the lines. The resistant parent (CM334) was the only
source of resistance at 5dpi. Heritability for resistance to individual isolates was 0.76,
0.74, 0.83, for isolates 12889, 13709 and OP97, respectively. Isolate OP97 at 5 days post
inoculation was the most virulent isolate and resistance to this isolate had the highest
heritability. Resistant loci were detected on chr 4 (13 cM) and 5 (29.8 cM) explaining 19
and 50% of the variation observed, respectively. An interaction between the two QTL
explained another 16% of the variation. Isolate 13709 had QTL located on chr 3 (124
cM), 5 (74 cM) and 6 (118 cM, P=0.05) explaining 14.5, 31, and 10% of the total
variation observed, respectively. Interactions between the QTL on chr 5 and that on chr 6
explained an additional 7%. There were no isolate-specific QTL detected for isolate
12889 at 5 days post inoculation.
Significant QTL were detected for fruit firmness, fruit shape and pericarp
thickness (Table 1.3). All QTL are significant at P=0.01 unless otherwise specified. One
QTL was detected for fruit firmness located on chr 12 at 55.4cM. This QTL explained
Table 1.3. Fruit phenotype QTL
QTL with additive effects

2

LOD R
Source Sig
4.486 0.298
EJ
***

Trait
Firmness

Chr.
12

Pos.
55.4

Marker
KS21041M02

Fruit shape

1
2
4
5
10

124
71.3
32.2
38
2.2

CAPS_CONTIG.816
CAPS_CONTIG.3755
KS20017D07
KS17024G04
KS26009E11

1.98
1.83
7.58
3.89
1.79

0.055
0.051
0.264
0.117
0.049

CM
CM
EJ
EJ
CM

*
*
***
***
*

Pericarp

3

114

KS17057E04

4.14

0.26

EJ

***

!$*!

Table 1.3 (cont’d)
QTL with epistatic effects
Trait

Chr.

Pos.

Fruit shape

P1*P4

124*32.2

Marker
KS20017D07 *
CAPS_CONTIG.816

LOD
1.72

R

2

Source

Sig

0.047 EJ*CM

*

Additive and epistatic effect QTL for fruit firmness and fruit shape. Chromosome and
genetic map position (position) of the marker. LOD value of the QTL, the percent of the
2
variation explained by the QTL (R ) and the donor parent of the allele (Source).
Significance of the QTL were determined by the general linear model implemented in
R/QTL at P=0.05 (*), P=0.01 (**), and P=0.001 (***).
approximately 29.8% of the variation within the population. Fruit shape QTL were
detected on chr 1 (P=0.05), 2 (P=0.05), 4, 5 and 10 (P=0.05) at positions 124, 71.3, 32.2,
38 and 2.2 cM, respectively. Together these QTL explained over 50% of the variation
observed. An epistatic interaction (P=0.05) between the QTL located on chr 1 and 4
explained an additional 4.7%. Pericarp thickness resulted in a single QTL chr 3 (114 cM)
that explained 26% of the variation. This QTL was tightly linked with a QTL for
resistance to isolate 13709 5dpi. QTL for fruit firmness did not show a tight linkage ("
10cM) with any of the disease QTL mapped.

DISCUSSION
Host resistance to P. capsici is an important component for disease management
in many areas of the world. Disease symptoms and the genetic factors controlling
resistance to the pathogen can vary greatly depending on the site of infection. In addition
to site-specific responses, isolate-specific interactions can be often host specific
((17,31,73,97)Foster and Hausbeck 2009; Quesada-Ocampo and Hausbeck 2010;
McGregor 2011, Candole 2012). Even resistant commercial peppers lines could succumb
!$+!

to highly virulent isolates when tested under greenhouse conditions (Foster and Hausbeck
2010). This combination of site-specific and isolate-specific responses makes breeding
for resistance to P. capsici incredibly challenging.
Breeding for all three symptoms of Phytophthora infection (foliar blight, root rot
and fruit rot) is important for a sustainable and effective resistance. Previously, studies
have mapped root rot resistance and foliar blight in multiple pepper populations, but not
fruit rot. Root rot and foliar blight are both estimated to be controlled by multiple QTL
and to exhibit isolate-specific responses. Root rot is the most common symptom seen in
the field, but under certain conditions, fruit rot can quickly decimate yields (8). Soilapplied systemic fungicides, a staple of any P. capsici management program, do not
provide protection to fruit.
The idea of early and late Phytophthora resistance was first suggested on pepper
roots by Pochard et al., who observed that roots exhibited an early response termed
receptivity, a late response termed stability and the rate of response called induciblity
(89). In our fruit study, we looked at early (3dpi) and late (5dpi) responses based
preliminary results of the earliest symptoms on commercial peppers and longest time to
cover the fruit. Three days post inoculation approximately half of the lines exhibited few
symptoms suggesting that partial early resistance was present in a 1:1 ratio in the
population, though multiple QTL were detected. Resistance at 5dpi was much less
common (21 individuals) and the only QTL identified were from the resistant parent.
These results at 5dpi contrast with previous results by Saini and Sharma (106) that
found resistance to be controlled by a single dominant trait. In this study, resistance to
Phytophthora fruit rot at 3 and 5dpi were both controlled by multiple genes of varying

!$,!

effects, with high overall heritability. In addition, the susceptible parent was also found to
contribute a positive effect on disease resistance during early stages of response. It
should be noted however, that as these fruit were evaluated under ideal conditions for
disease that some of the lines evaluated in this study with partial resistance may be more
resistant under field conditions or as the fruit matures. Studies have shown that resistance
to P. capsici increases for fruit and plants as tissues mature (9,36,53,61). Fruit rot
resistance appeared to be controlled by a single dominant gene in ‘Waxy Globe’, but in
CM334 the trait is quantitatively inherited and highly influenced by the environment. The
heritability of fruit rot resistance at 5dpi was high, and the detection of major QTL
controlling resistance may make marker-assisted selection useful.
Fruit traits evaluated also showed evidence of quantitative inheritance, consistent
with previous studies (19,124,126). QTL for fruit firmness did not show a tight linkage ("
10cM) with any of the disease QTL mapped. The fruit shape QTL on chr 5 was linked
with a major effect QTL for resistance to OP97 5dpi (54% of the variation) and a
moderate effect on fruit shape (~12%). The fruit shape QTL on chr 10 was a minor QTL
(<10%) for both fruit rot resistance and fruit shape. This was consistent with the previous
study that detected a small yet significant correlation between fruit shape and disease for
at least one isolate (Naegele and Hausbeck (in review).) Pericarp thickness also had
potential linkage with a disease resistance QTL with disease. This pericarp thickness
QTL was within 10cM of a resistance QTL explaining 18% of the disease variation 5dpi.
However, the linked QTL was only found in disease resistance to a particular isolate, and
may not be useful when breeding for resistance to multiple isolates.

!%-!

Most QTL identified 3 and 5dpi were located on chromosome 5, a known hot spot
for Phytophthora resistance genes in several solanaceous species including a major gene
for root rot resistance to P. capsici (43,112). The quantitative inheritance of fruit rot is
consistent with previous studies on root rot and foliar blight in peppers (12,76,80). The
resistant parent used in this study, CM334, though more tolerant than Early Jalapeno, was
not immune to Phytophthora fruit rot under our conditions, and may not be the best
source of fruit rot resistance. In addition, the cultivated parent (Early Jalapeno)
contributed two loci on chromosome 6 to resistance 3dpi, making commercial cultivars a
potential source of early resistance. In this study, the QTL effects are likely to be slightly
overestimated due to the limited number of individuals evaluated in this population
(Broman 2009), but provide a good first approximation. Other sources of resistance may
have more complete resistance and more evaluation of lines is needed. For the QTL that
were detected, further work with larger populations and more isolates will be needed to
confirm the QTL in different genetic backgrounds and identify additional ones.

!%$!

CHAPTER 2: GENETIC DIVERSITY, POPULATION STRUCTURE, AND
RESISTANCE TO PHYTOPHTHORA CAPSICI OF A WORLDWIDE
COLLECTION OF EGGPLANT GERMPLASM
ABSTRACT
Eggplant (Solanum melongena L.) is an important Solanaceous crop with high
phenotypic diversity and moderate genotypic diversity. The objectives of this study were
to genetically characterize a worldwide collection of eggplants evaluated for resistance to
Phytophthora capsici fruit rot. Ninety-nine genotypes of eggplant germplasm (species,
landraces and heirloom cultivars) from 32 countries and five continents were evaluated
for genetic diversity, population structure, fruit shape, and disease resistance to
Phytophthora fruit rot. Fruit from each line were measured for fruit shape and evaluated
for resistance to two Phytophthora capsici isolates seven days post inoculation. One line
(PI 413784) was completely resistant to both isolates evaluated, while several others
exhibited isolate-specific resistance. Partial resistance to Phytophthora fruit rot was found
in accessions from all three eggplant species (S. incanum, S. linneanum and S.
melongena). Genetic diversity and population structure were assessed using 22
polymorphic simple sequence repeats (SSRs). Genetic analyses using the program
STRUCTURE indicated the existence of four genetic clusters within the eggplant
collection. Population structure was detected when eggplant lines were grouped by
eggplant species, continent of origin, country of origin, fruit shape and disease resistance.

!%%!

INTRODUCTION
Cultivated eggplant, Solanum melongena L., is a high-value vegetable commodity
in Europe and Asia. China and India are the major producers with 27.7 and 11.9 million
tons per year, respectively [2011; FAO]. Eggplant is the third most important
Solanaceous crop worldwide, and fourth most important in the U.S., after potato and
tomato [2011; FAO]. In the U.S., eggplants are a minor crop grown for specialty markets
with an approximate production of 62 thousand tons annually [2011; FAO].
Unlike most other cultivated Solanaceous crops (tomatoes, peppers, and potatoes),
eggplants are an Old World species. Domestication of the cultivated eggplant is thought
to have occurred in Asia as early as 59 B.C. (52,75,118). Since that time, it has been
transported and cultivated around the world (93,121). Centers of diversity for eggplant
are Asia, Europe and Africa. Studies have indicated that the progenitors of domesticated
eggplant (S. melongena) originated in Africa and were derived from the closely related
Solanum incanum (part of the S. melongena complex) and S. linnaeanum (121). Both S.
incanum and Solanum linnaeanum can form partially fertile hybrids with S. melongena
making them potential sources for desirable traits such as abiotic and biotic disease
resistance (24,25,46). Wild relatives have traditionally been a good source of disease
resistance for cultivated species that exhibit lower genetic diversity (26,56,108).
Heirloom varieties and landrace accessions might also harbor resistance, and are often
more similar to modern cultivated varieties than wild species, making them a good source
for desirable traits (78).
Phytophthora fruit rot caused by Phytophthora capsici L., an oomycete, is a
disease that affects multiple solanaceous species including eggplant, pepper, and tomato

!%&!

(31,32,40,95,97). In the field, chemical management is expensive and provides limited
protection against Phytophthora fruit rot in eggplants, which is the most common
Phytophthora-induced disease in eggplants (37). Host resistance, an important part of a
successful, sustainable management program, is not available for management of
Phytophthora fruit rot in eggplants and currently, no known lines or cultivars are resistant
to P. capsici. Partial fruit rot resistance to P. capsici has been identified in other
solanaceous species such as peppers and tomatoes (Naegele et al. unpublished; Granke et
al. unpublished), but to our knowledge this has not been evaluated in eggplant.
The linkage or correlation of resistance/susceptibility to insects and pathogens
with agronomic traits of interest is not uncommon in plants (22,35,74). Correlations with
resistance to Phytophthora fruit rot and fruit sugar content and exocarp thickness have
been found in cucurbits (74). In peppers, pericarp thickness and pungency were not
correlated with resistance, but fruit shape was positively associated with increased
susceptibility to Phytophthora fruit rot (44).
In addition to disease resistance, fruit shape is an important attribute for each
cultivar and many studies have been performed to identify the genetic basis of fruit shape
in the Solanaceae (79,94,103,113,123,126). Size, shape and color vary greatly between
eggplant market classes, and it will be important to maintain this phenotypic diversity
when incorporating disease resistance (90,93). This phenotypic diversity does not always
translate to high levels of genetic diversity (72,93). Modern varieties of eggplant often
have lower genetic diversity, and new traits are often bred into commercial varieties from
landraces or wild relatives with higher genetic diversity (78). The characterization of
genetic diversity is important for maintenance and utilization of germplasm resources

!%'!

(wild, landrace, heirloom, breeding lines and cultivars), and the development of core
collections (21,52). Genetic bottlenecks (domestication, selection of lines by market
class, etc.) have limited the variability existing within cultivated lines, and eggplant has
had an additional bottleneck as a result of domestication outside of its center of origin
(52).
Population structure analysis has recently been gaining popularity as a method to
understand and visualize spatial and temporal differences between subpopulations
(23,33,55,97,107). Information on population structure can provide insight about
connections between phenotypic variation and the distribution of genetic diversity.
Population structure should also be taken into account when testing and incorporating
desirable traits. If population structure is present in materials being evaluated for
association mapping, spurious associations may be made between a particular genotype
and the trait of interest (90,119). Many studies have looked at the genetic diversity of
eggplants within specific countries or regions, and a recent study compared genetic
differentiation and structure in three centers of diversity; however, no studies have looked
at diversity and population structure in a global collection of eggplants
(4,6,34,52,59,75,78,90,93).
We evaluated fruit shape, Phytophthora fruit rot resistance, genetic diversity and
population structure of a diverse collection of eggplant germplasm using 22 simple
sequence repeats (SSRs). Our objectives were to evaluate a worldwide collection of
eggplants for population structure and genetic diversity, and to determine if the
population structure is associated with fruit shape or resistance to Phytophthora capsici.
These results provide an initial basis for understanding the worldwide population

!%(!

structure of eggplants for breeding and conservation, and its relationship with disease
resistance and fruit shape.
MATERIALS AND METHODS
Ninety-five accessions of eggplants (S. melongena), 3 accessions of S.
linnaeanum and 1 accession of S. incanum were obtained from the United States
Department of Agriculture Germplasm Resource Information Network (arsgrin.usda.gov), Dr. J. Prohens (Universidad de Technologia de Valencia), and the INRA
(French National Institute for Agricultural Research) (Table 1.1).
Table 2.1. Eggplant germplasm used for the study of morphological and molecular
variation
Species
ID Accession
Plant ID
Country
Source
S. incanum
191 PI 500922
Zambia
USDA
S. linnaeanum 174 PI 388846
WL-74
Italy
USDA
S. linnaeanum 175 PI 388847
WL-85
Italy
USDA
S. linnaeanum 182 PI 420415
52
Colombia
USDA
S. melongena 101
C-S-16
Spain
Dr. J. Prohens
S. melongena 102
H15
Spain
Dr. J. Prohens
S. melongena 103 IVIA-371
Spain
Dr. J. Prohens
MM 108
S. melongena 104
bis
France
INRA
Berengena larga
S. melongena 105 MM 114
negra
Spain
INRA
S. melongena 106 MM 1171 Large Santa Olalla
Spain
INRA
S. melongena 107 MM 1363
Costa Rica
INRA
S. melongena 108 MM 1364
Costa Rica
INRA
S. melongena 109 MM 1365
Guatemala
INRA
S. melongena 110 MM 141
Violette d'Avignon
France
INRA
S. melongena 111 MM 1750
Listada di Gandia
Spain
INRA
S. melongena 112 MM 346 Berengena redonda
Spain
INRA
Noire de
S. melongena 113 MM 39
Chateaurenard
France
INRA
S. melongena 114 MM 522 Waimanolo long B1
USA
INRA
Violette de
S. melongena 115 MM 56
Toulouse
France
INRA
S. melongena 116 MM 61
Zebrina
Spain
INRA
S. melongena 117 MM 64
Ronde de Valence
France
INRA
Monstrueuse de
S. melongena 118 MM 69
New York
USA
INRA
!%)!

Table 2.1 (cont’d)
S. melongena 119
S. melongena 120
S. melongena

121

S. melongena
S. melongena
S. melongena
S. melongena
S. melongena
S. melongena
S. melongena
S. melongena
S. melongena
S. melongena
S. melongena
S. melongena
S. melongena
S. melongena
S. melongena
S. melongena
S. melongena
S. melongena
S. melongena
S. melongena
S. melongena
S. melongena
S. melongena
S. melongena
S. melongena
S. melongena
S. melongena
S. melongena
S. melongena
S. melongena
S. melongena
S. melongena
S. melongena
S. melongena

122
123
124
125
126
127
128
129
130
131
132
133
134
135
136
137
138
140
141
142
143
144
145
146
147
148
149
150
151
152
153
154
155
156

MM 91
Black Beauty
Grif 1276
46B
Grif
New Orleans
14182
Market
Grif
Hastings imp purple
14186
thornless
PI 102727
No. 202
PI 105346 Lao Lai Hei Chieh
PI 115505
Giant of Benares
PI 140446
5917
PI 140456
7015
PI 141968
No. 1
PI 143410
Badenjan
PI 169641
1448
PI 169650
2259
PI 171851
6753
PI 175914
9043
PI 179500
9877
PI 179997
10598
PI 181896
Aleppo 3
PI 181963
Homs 21
PI 193599
Long Violet
PI 199516
M 19
PI 200881
PI 204731
PI 213193
M-57/29
PI 217962
Banjal Bemba
PI 223844
PI 230333
Kairyo-onaga
PI 230334
Kitta Horyo
PI 230335
Taiwan-naga
PI 232078
Kopek
PI 232079
Mofale
PI 233916
PI 234632 Early Round Purple
PI 241506
Badanjan
PI 249570
Makhua Proh
PI 256077
No.1
PI 263727
Rosita

S. melongena
S. melongena
S. melongena
S. melongena
S. melongena

157
158
159
160
161

PI 267104
PI 269600
PI 276104
PI 286099
PI 286100

Cylinder A-132
423
Motale
No. 62-46-2
No. 62-48-2
!%*!

USA
Thailand

INRA
USDA
USDA

USA
USDA
USA
Uzbekistan
China
India
Iran
Iran
China
Iran
Turkey
Turkey
Turkey
Turkey
Iraq
India
Syria
Syria
Ethiopia
Greece
Afghanistan
Turkey
Greece
Pakistan
Philippines
Japan
Japan
Japan
South Africa
South Africa
El Salvador
South Africa
Iran
Thailand
Afghanistan
Puerto Rico
Former Soviet
Union
Pakistan
South Africa
USA
USA

USDA
USDA
USDA
USDA
USDA
USDA
USDA
USDA
USDA
USDA
USDA
USDA
USDA
USDA
USDA
USDA
USDA
USDA
USDA
USDA
USDA
USDA
USDA
USDA
USDA
USDA
USDA
USDA
USDA
USDA
USDA
USDA
USDA
USDA
USDA
USDA
USDA
USDA

Table 2.1 (cont’d)
S. melongena 162
S. melongena 163
S. melongena 164
S. melongena 165
S. melongena 166
S. melongena 167
S. melongena 168
S. melongena 169
S. melongena 170
S. melongena 171
S. melongena 172
S. melongena 173
S. melongena 176
S. melongena 178
S. melongena 179
S. melongena 180
S. melongena 181
S. melongena 183

PI 290467
PI 290469
PI 304839
PI 320501
PI 320504
PI 320509
PI 349612
PI 351129
PI 358232
PI 358242
PI 358244
PI 368822
PI 391646
PI 413782
PI 413783
PI 413784
PI 419198
PI 441908

S. melongena

184 PI 452122

S. melongena
S. melongena
S. melongena
S. melongena
S. melongena
S. melongena
S. melongena
S. melongena
S. melongena
S. melongena
S. melongena
S. melongena
S. melongena
S. melongena
S. melongena
S. melongena

185
186
187
189
190
192
193
194
195
196
198
199
200
201
202
203

Lungi de Impant
Cu-e-da-juan
G2562
24
28
35
Terongglatik
Kurume Long
Dolg
Morska Pata
Renski dolg
Sredno Dolg
Liu-ye-ch'ieh
22-73
3-73
13-73
Tsu Yang
BGH 5008
Lunga Violetta di
Romagna
Tonda di
Manfredonia
Neznyj 36

PI 452123
PI 462370
PI 470273
PI 478390
O 81
PI 491192
Kemer
PI 560903
Six Leaves
PI 561139
37
PI 561140
36
PI 593748
56A
PI 593806
171
PI 593885
314
PI 595220
Gator
PI 600912
Little fingers
PI 606714
Pompano market
PI 639121 Puerto Rican beauty
PI 639122
Blackee

Hungary
Hungary
Brazil
Canada
Canada
Canada
Indonesia
Japan
Macedonia
Macedonia
Macedonia
Macedonia
China
Cote D'Ivoire
Burkina Faso
Burkina Faso
China
Brazil

USDA
USDA
USDA
USDA
USDA
USDA
USDA
USDA
USDA
USDA
USDA
USDA
USDA
USDA
USDA
USDA
USDA
USDA
USDA

Italy
USDA
Italy
Soviet
Indonesia
China
Turkey
China
Kazakhstan
Kazakhstan
Thaliand
Thailand
Thailand
USA
USA
USA
Puerto Rico
USA

USDA
USDA
USDA
USDA
USDA
USDA
USDA
USDA
USDA
USDA
USDA
USDA
USDA
USDA
USDA

Seeds were planted into 72-cell trays containing a soilless peat mixture (Suremix
Michigan Grower Products, Inc. Galesburg, MI) in a polyethylene greenhouse (MSU
Horticulture Teaching Farm, East Lansing, MI). Eight weeks after planting, seedlings
!%+!

were transplanted to the MSU Plant Pathology Farm (East Lansing, MI). Individual lines
were planted into single plots. Each individual line was established in 3 m long plot and
12 lines were planted per row. Within rows, plants were spaced 0.45 m apart. Rows were
spaced 2.4 m apart, covered with black plastic mulch, and grown according to local
practices. Immature eggplant fruit of marketable size were hand harvested and brought to
the lab for inoculation and evaluation.
Two P. capsici isolates were selected from the long-term collection of Dr. Mary
K. Hausbeck at MSU. Isolates were characterized by host of origin, mefenoxam
sensitivity [insensitive (I) or sensitive (S)] and mating type (A1 or A2). Isolate 12889
(pepper, I, A1) and isolate OP97 (cucumber, S, A1) were maintained on unclarified V8
agar at 25 °C under constant light. Prior to inoculations, isolates were activated by
inoculating and recovering each isolate from an individual pepper fruit to ensure
virulence.
For inoculation, a single 6mm-diameter plug from an actively growing P. capsici
isolate on V8 agar was placed, mycelium side down onto a non-wounded eggplant fruit
surface-disinfested in 10% bleach for 5 min. Control eggplants were inoculated with a
single 6mm-diameter sterile plug of agar. Plugs were covered with a sterile
microcentrifuge tube and affixed into place with petroleum jelly. Eggplants were placed
into a humidity chamber consisting of an aluminum pan with a ring of moistened paper
towel around the edge, covered with plastic wrap, sealed with tape and kept under
constant light at room temperature (25 °C). Three fruit from each eggplant line were
evaluated per isolate. The experiment was performed three times. An experiment
replicate included three fruit for each isolate of every line evaluated in a completely

!%,!

randomized design (CRD) blocked by isolate. One line (PI 500922) was repeated only
one time for a total of two experimental replicates of three fruit per isolate due to poor
fruit set. Two control fruit were inoculated with a sterile plug of V8 agar for each line.
Eggplant fruit were evaluated for disease severity seven days after inoculation.
Fruit were evaluated on the following progressive scale based on percentage of fruit
diseased to account for differences in fruit size: 0= no visible symptoms, 1=<25% of the
fruit was symptomatic, 2= 25% to <50%, 3= 50% to <75%, 4= 75% or greater percent
symptomatic area (Fig. 2.1).
Figure 2.1. Eggplant Phytophthora fruit rot disease rating scale shown on various
eggplant genotypes: 0= no visible symptoms, a rating of 1=<25% symptomatic area,
25%>2<50%, 50%>3<75%, and a rating of 4!75% symptomatic area of the fruit. Visible
pathogen growth was assessed as 0=absent, 1=present. (For interpretation of the
references to color in this and all other figures, the reader is referred to the electronic
version of this dissertation).

Isolations were performed on 10% of symptomatic fruit by peeling back the
external layer of the fruit and plating three small portions of fruit at the disease margin
onto V8 agar plates amended with benoymyl, ampicillin, PCNB and mefenoxam [50]. P.
capsici was identified using morphological characteristics according to Waterhouse and
isolate mefenoxam sensitivity was confirmed by transferring the recovered isolates to V8
plates amended with 100ppm mefenoxam (120).
!&-!

Ten immature fruit of marketable size collected from each line were measured for
maximum length (cm) and midpoint width (cm) using a hand caliper. Fruit shape was
calculated as the ratio of maximum length to midpoint width for each line. Fruit shape
ratios were rounded to the nearest whole number. Values between 0 and 1 were
considered round, 2-3 were considered oval, 4-5 were semi-elongate and greater than 5
were considered elongate.
Mean values for disease ratings for each line were estimated using the PROC
MEANS function of SAS software v9.3 (SAS Institute Cary, NC). Significant differences
between disease values (ratings) for lines and isolates were estimated using the PROC
MIXED function of SAS software. Significant differences were detected between
experiment replicates and each replicate was analyzed separately using Fisher’s LSD test
(P=0.05). Line-by-isolate interactions were calculated using the ANOVA slice option of
PROC MIXED when P"0.05. Lines with a consistent disease mean value of 2 or greater
in each run of the experiment were considered susceptible, with a consistent mean value
<2 were termed moderately susceptible, lines with a consistent mean value <1 were
moderately resistant, and lines with a mean value = 0 were resistant. Significant
differences for pathogen growth were estimated using the PROC GLIMMIX function of
SAS at P=0.05.
Fruit shape significant differences between lines and countries were calculated
using the PROC mixed function of SAS software v9.3. Countries represented by less than
four lines were excluded from analyses. Unequal sample sizes among countries were
accounted using the Kenward-Rogers degrees of freedom option implemented in SAS
software. Line mean values for fruit shape, length and width were calculated using the

!&$!

lsmeans statement of SAS software. Correlations between fruit shape parameters and
disease susceptibility were estimated using the PROC CORR function of SAS. Disease
susceptibility correlations were evaluated for each isolate and experimental replicate
separately.
Genomic DNA was extracted from young green leaves of eggplants using the
Nucleo Spin II DNA extraction kit (Machery-Nagel Germany, CAT#740770) according
to the manufacturer’s instructions. DNA was normalized to 5 ng/ul using the NanoDrop
ND 1000 spectrophotometer and NanoDrop 2.4.7c software (NanoDrop Technologies
Inc., Wilmington, DE).
One hundred ninety-two primers from previously published SSR markers (3, 52,
solgenomics.org) or designed (Primer 3 http://primer3.sourceforge.net/) from putative
Solanaceae defense-related genes (NCBI ncbi.nlm.nih.gov) were tested against a subset
of the eggplant collection to identify polymorphic markers. Reactions were performed in
15 ul total volume and contained 1 ul DNA, and 0.15 ul GoTaq (Promega Corporation
Madison, Wisconsin), 0.9 ul 25uM MgCl2, 0.3 ul dNTPs, and 0.6 ul each of forward and
reverse primers (Integrated DNA Technologies, Inc.), with 8.45 ul ddH2O. PCR reactions
were performed in a programmable thermal cycler (Eppendorf, Westbury, NY) using the
program: initial denaturation, 94 C (3 min) followed by 35 cycles at 94 C (30s), 60 C
(30s) and 72 C (1 min), with a final extension step of 10 min at 72 C. PCR products were
analyzed by electrophoresis in 4% (wt/vol) agarose gel in 1x Tris-borate-EDTA buffer,
stained with ethidium bromide (5 ug/ml) for visualization and compared to a 100-bp
ladder (Invitrogen Life Technologies Burlington, ON Canada) to determine amplicon

!&%!

sizes. SSR markers identified as polymorphic in the population were used for genetic
diversity, population structure and trait associations.
Genetic diversity was estimated using Powermarker v3.25 (71) and significance at
each locus was determined with 1000 permutations using the Exact test; overall genetic
diversity was estimated using the Mantel test as implemented in Powermarker.
Population structure of the germplasm was analyzed using STRUCTURE v2.3.4
(92). Following preliminary analyses, burnin length, MCMC chain replication and
lambda were selected to be 200,000, 500,000 and 1.52, respectively. Population number
(k) was determined empirically by comparing posterior distribution likelihoods
independently among 3 independent runs of K=1 to 20 as described by Evanno et al. (27).
Data included 22 polymorphic SSR and were analyzed using the admixture model and
correlated allele frequencies without previous population information (28,92). Fst
significance between populations was determined using 1000 bootstrap replicates as
implemented in Powermarker.
Visualization of the resulting Q (proportion of membership) of each individual
into predefined categories (country, continent, species, disease susceptibility and fruit
shape) was generated using the Population Sorting Tool (PST) in R [19,58, J.J. Morrice,
unpublished]. Individuals with Q!0.6 membership in a single subpopulation were labeled
as such. Individuals with Q<0.6 membership in a single subpopulation were considered
admixed. Significance of population structure by continent, country, species, resistance to
P. capsici (12889 and OP97), and fruit shape was estimated using the population
differentiation test implemented in Powermarker. Significance at each locus and overall
was determined using 1000 permutations. Countries represented by less than four

!&&!

individuals were excluded from analyses. Significance of pairwise Fst differentiation was
based on 2.5 and 97.5% confidence intervals based off of 1000 bootstrap replications.

RESULTS
Significant differences between experimental replicates indicated the effect of
environmental variability on fruit disease susceptibility was high. In each repetition of the
experiment, there were significant differences among plant lines (P<0.0001). No
significant differences were found between isolates in any replicate of the experiment (P=
0.32, P= 0.43, and P= 0.43). The interaction between line and isolate was significant for
each replicate (Replicate 1: P= 0.0008; Replicate 2: P< 0.0001; and Replicate 3 P<
0.0001) of the experiment. Differences in pathogen growth (absence/presence) and the
interaction between pathogen growth and line were not significant in any replicate
(approximately P=1.0 for each). The majority of the lines 89% and 87% evaluated were
susceptible at 7 dpi (days post inoculation) to isolates OP97 and 12889, respectively
(Table 2.2).
Table 2.2. Fruit shape parameters and mean disease ratings for each isolate overall and
per individual experiment.
a

Species
S. incanum
S. linnaeanum
S. linnaeanum
S. linnaeanum
S. melongena
S. melongena
S. melongena
S. melongena
S. melongena
S. melongena
S. melongena

Accession
PI 500922
PI 388846
PI 388847
PI 420415
C-S-16
Grif 1276
Grif 14182
Grif 14186
H15
IVIA-371
MM 108 bis

Mean
12889 OP97
MR
MS
MR
S
MR
S
S
S
S
S
MS
R
S
S
S
S
R
S
S
S
S
S
!&'!

b

Fruit
Ratio Length Width
1.03
2.6
2.6
1.03
2.5
2.4
1.07
2.0
1.9
1.06
2.1
2.1
5.59
23.0
4.1
1.14
4.9
4.3
2.59
15.0
6.1
1.75
13.5
7.8
1.93
11.8
6.2
2.08
15.0
7.3
5.23
21.5
4.2

Table 2.2 (cont’d)
S. melongena
S. melongena
S. melongena
S. melongena
S. melongena
S. melongena
S. melongena
S. melongena
S. melongena
S. melongena
S. melongena
S. melongena
S. melongena
S. melongena
S. melongena
S. melongena
S. melongena
S. melongena
S. melongena
S. melongena
S. melongena
S. melongena
S. melongena
S. melongena
S. melongena
S. melongena
S. melongena
S. melongena
S. melongena
S. melongena
S. melongena
S. melongena
S. melongena
S. melongena
S. melongena
S. melongena
S. melongena
S. melongena
S. melongena
S. melongena
S. melongena
S. melongena
S. melongena
S. melongena
S. melongena

MM 114
MM 1171
MM 1363
MM 1364
MM 1365
MM 141
MM 1750
MM 346
MM 39
MM 522
MM 56
MM 61
MM 64
MM 69
MM 91
PI 102727
PI 105346
PI 115505
PI 140446
PI 140456
PI 141968
PI 143410
PI 169641
PI 169650
PI 171851
PI 175914
PI 179500
PI 179997
PI 181896
PI 181963
PI 193599
PI 199516
PI 200881
PI 204731
PI 213193
PI 217962
PI 223844
PI 230333
PI 230334
PI 230335
PI 232078
PI 232079
PI 233916
PI 234632
PI 241506

S
S
S
S
S
S
S
S
S
S
MS
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
MS
S
S
S
S
S
S
S
S
S
S
S
S
S
MS
S
MS

S
S
S
S
MS
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
MS
S
S
S
S
S
S
S
S
S
S
S
S
S
S
!&(!

6.57
2.81
5.05
3.02
1.87
4.67
2.49
1.31
5.37
8.03
2.42
1.97
1.16
1.34
1.92
2.44
1.17
1.72
1.77
3.48
4.46
1.35
3.78
4.66
4.31
2.92
3.64
3.34
1.91
3.99
1.84
1.74
3.82
2.73
1.07
3.26
2.89
7.15
6.84
7.43
4.01
2.36
2.37
0.93
2.73

22.2
15.1
25.8
17.8
14.6
23.8
17.5
11.4
22.9
26.1
16.3
11.5
10.0
11.4
14.2
14.8
10.3
11.3
12.6
21.9
19.9
10.7
19.3
19.2
17.8
14.1
16.0
15.9
12.5
16.0
11.6
14.2
22.1
18.1
9.8
15.4
14.8
25.6
21.2
26.2
18.2
13.1
13.2
7.9
16.4

3.4
5.4
5.1
6.0
7.9
5.1
7.1
8.7
4.3
3.3
6.8
5.9
8.7
8.8
7.5
6.1
8.9
6.6
7.1
6.3
4.5
7.9
5.2
4.2
4.1
4.8
4.4
4.8
6.6
4.0
6.5
8.8
5.9
7.6
9.2
4.8
5.3
3.6
3.1
3.5
4.6
5.6
5.6
8.7
6.1

Table 2.2 (cont’d)
S. melongena
S. melongena
S. melongena
S. melongena
S. melongena
S. melongena
S. melongena
S. melongena
S. melongena
S. melongena
S. melongena
S. melongena
S. melongena
S. melongena
S. melongena
S. melongena
S. melongena
S. melongena
S. melongena
S. melongena
S. melongena
S. melongena
S. melongena
S. melongena
S. melongena
S. melongena
S. melongena
S. melongena
S. melongena
S. melongena
S. melongena
S. melongena
S. melongena
S. melongena
S. melongena
S. melongena
S. melongena
S. melongena
S. melongena
S. melongena
S. melongena
S. melongena
S. melongena
a
b

PI 249570
PI 256077
PI 263727
PI 267104
PI 269600
PI 276104
PI 286099
PI 286100
PI 290467
PI 290469
PI 304839
PI 320501
PI 320504
PI 320509
PI 349612
PI 351129
PI 358232
PI 358242
PI 358244
PI 368822
PI 391646
PI 413782
PI 413783
PI 413784
PI 419198
PI 441908
PI 452122
PI 452123
PI 462370
PI 470273
PI 478390
PI 491192
PI 560903
PI 561139
PI 561140
PI 593748
PI 593806
PI 593885
PI 595220
PI 600912
PI 606714
PI 639121
PI 639122

S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
MS
R
S
R
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S

S
S
MS
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
R
MR
R
MS
MS
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S

1.53
3.07
1.95
3.93
1.71
2.22
5.81
6.45
3.34
2.16
2.85
2.18
4.58
2.46
1.44
5.61
4.59
2.16
5.36
3.18
5.33
0.79
0.46
0.69
5.63
0.83
5.79
1.29
1.15
3.30
0.75
4.95
0.95
2.94
3.48
2.65
3.79
1.12
2.70
4.58
2.40
1.96
1.60

10.0
16.1
12.8
18.8
11.6
15.6
25.3
28.1
21.0
14.9
17.5
15.5
29.1
17.1
7.4
26.5
22.4
14.5
24.7
18.2
25.8
1.2
2.0
4.1
24.3
4.3
23.8
12.2
12.0
15.9
7.1
22.1
8.2
16.2
16.2
15.2
15.9
6.6
12.5
15.3
13.3
14.0
10.8

5.8
5.3
8.0
4.9
7.1
7.0
4.5
4.7
6.3
7.0
6.2
7.2
6.6
6.9
5.1
4.7
4.9
7.2
4.7
5.8
8.2
1.5
4.4
5.9
4.4
5.2
4.1
9.6
10.9
4.9
9.5
4.5
8.7
5.6
4.7
5.8
4.2
6.0
4.6
3.4
5.6
7.3
6.7

Mean disease rating across all experimental replicates for each isolate, 12889 and OP97
Mean fruit parameters for ratio (fruit length: fruit width), length (cm), and width (cm)
!&)!

Symptoms included brown discoloration of the fruit and watersoaking, with
occasional external mycelial growth (Fig 2.1). Eggplant accession PI 413784 was the
only line completely resistant to both isolates tested. Susceptibility to one isolate did not
always result in susceptibility to the other isolate. Lines PI 413782 and Grif 1276 were
resistant (rating =0) to isolate OP97. PI 413783 was moderately resistant (rating <1) to
isolate OP97. The single S. incanum line, PI 500922, and S. melongena lines PI 441908,
MM1365, PI 193599, PI 263727 and PI 419198 were moderately susceptible (rating <2)
to isolate OP97. Eggplant lines H15 and PI 441908 were resistant to isolate 12889. Two
of the S. linnaeanum lines, PI 388847 and PI 388846, and the S. incanum accession PI
500922 were moderately resistant to isolate 12889. Lines PI 181896, PI 233916, MM 56,
and PI 413783, and Grif 1276 were moderately susceptible to isolate 12889.
Phytophthora capsici isolates were successfully recovered from diseased fruits and
mefenoxam sensitivity was confirmed for each isolate (data not shown). Fruit shape and
size varied considerably in the population (Fig 2.2).
Figure 2.2. Fruit size and shape differences between eggplants. S. incanum (left) and S.
linnaeanum (right) fruit (A), S. melongena fruit (B) and S. linnaeanum and S. melongena
fruit varying in shape, size and color (C). U.S. quarter used for size reference.

!&*!

S. melongena accessions had fruit shape ratios ranging from 1 (round) to 8 (elongate).
The wild species evaluated (S. linnaeanum and S. incanum) both had a fruit shape ratio of
approximately 1 (round) with fruit "3 cm (Table 2.2). Solanum melongena line PI
413783 had the lowest fruit shape ratio (0.46) and line MM 522 had the highest fruit
shape ratio (8). When evaluated by country, S. melongena fruit from Japan had the
highest length:width ratio indicating fruits were slender and elongated. Fruit from
Thailand had the lowest fruit shape ratio, indicating fruits were more round. Fruit length
and width also varied greatly between countries. Fruit from China were the widest and
Japan the narrowest. Fruit from Japan were also the longest and fruit from Thailand were
the shortest (Table 2.3). No consistent correlations were detected between fruit shape
ratio and width parameters and disease susceptibility for either isolate.
Table 2.3. Solanum spp. fruit shape, width and length variation between countries of
origin.
Fruit
a
b
c
d
Category
Shape
Width (cm)
Length (cm)
China
3.0 cd
7.4 a
15.9 de
France
3.8 b
5.8 cd
18.9 b
Iran
2.3 de
6.9 ab
15.4 de
Italy
3.5 bc
6.8 abc
18.0 bcd
Japan
6.8 a
3.7 e
24.9 a
Macedonia
3.8 b
5.6 cd
20.0 b
S. Africa
2.4 de
6.5 abc
13.7 ef
Spain
3.1 c
6.0 bc
15.9 d
Thailand
2.2 d
5.6 cd
11.8 f
Turkey
3.9 b
5.1 d
18.4 bc
USA
3.6 bc
5.7 cd
16.9 cd
a
Categories with less than five individuals representing a country were not included in
analyses
b
Mean fruit shape calculated as the ratio of fruit length to fruit width
c

d

Mean fruit width at midpoint measured in cm
Mean fruit length from peduncle to blossom end measured in cm

!&+!

2

Fruit length was positively correlated with fruit susceptibility (R = 0.20-0.35, P<0.05)
for both isolates in each experimental replicate (Table 2.4).
Table 2.4. Correlation of Solanum spp. fruit characteristics with disease susceptibility
b

Category
Length
Width
Shape
a

a

12889 (1)

Pearson Correlation Coefficient
12889 (2) 12889 (3) OP97 (1)
OP97 (2)

OP97 (3)

0.25*
0.32**
0.12

0.22*
0.08
0.18

0.27**
0.03
0.2*

0.2*
0.07
0.15

0.36***
0.17
0.27**

0.22*
0.15
0.14

Mean fruit phenotype category for each line correlated against disease susceptibility;
Length = fruit length in cm from peduncle to blossom end, Width = fruit width in cm at
midpoint between peduncle and blossom end, Shape = fruit shape as the length:width
ratio
b
Correlation coefficient value according the Pearson’s Correlation Coefficient of each
category with isolate 12889 or OP97 at biological replicates 1, 2 and 3; *, **, and ***
indicate that values were significant at P=0.05, 0.01 and 0.001, respectively.

The 192 SSRs evaluated yielded 22 polymorphic markers that were used for
characterizing and evaluating genetic diversity of the eggplant collection (Table 2.5). A
total of 83 alleles were detected among the 22 SSRs, ranging from 2 to 7 alleles per locus
with an average allele diversity of 3.8 alleles per locus. The mean genetic diversity index
of the collection was 0.49 ranging from 0.03 (T0633) to 0.76 (CSM31) (Table 2.5). The
mean polymorphism information content (PIC) value was 0.42 and individual markers
ranged from 0.03 to 0.71 for the population. The highest PIC value was 0.35 in PI 290467
and the lowest PIC value was 0.085 in Grif 1276. Genetic diversity was equally
distributed within continents (0.47-0.51), and pairwise Fsts indicated low to moderate
genetic differentiation between continents (0.00 – 0.11) (Table 2.6). Genetic diversity

!&,!

Table 2.5. Polymorphic primers evaluated against 99 eggplant lines
SSR
BM61461
GPMS203
CB164833
T0633
CA516334

a
b

Forward sequence
CTCATTACCACTTCATACAAAACAG
CACCAACACATCTTTTTCAACC
CGGGCAGGTGCTATTATAAAAC
GATGGGCTATGCTTGCTGTT
ACCCACCTTCATCAACAACC

Reverse sequence
TGCAGTAGGTGTTGCTACGG
ATAATAGTGGTTGCGGCGAC
CGGCCGAGGTACAAGCC
ACATCCCCAATGTTGTTGTG
ATTTGTGGCTTTTCGAAACG
AACGTTGAAAAATAAAGTAAGC
AAG
TTTGCCCGCATTATGTAAATC
GAACAAAACATGCCCTACTGTA
GGAA
TCTTTTCTTGTATTGGCGGCTAA
ATTC
CCATACGGACGTTGTCCTCT
TATGCCCCTCCTGTGATAGC

GPMS178
GATTTTTGACATGTCACATTCATG
GP1102
GAACCCTTCATTCCTGTATGT
C2_At5g34
850
AGTGAAGTGGCTACATCCAAAATCTC
C2_At1g69 AGCTCTATTCATTTAAAACTAGTCCTCA
210
T
AF348141
CCTTACGGGGAAAACCTAGC
CAMS362
CCCCTTCTGACCTTGATTGA
GO496268.
1
CGTTGCCTGTTTACCAACCT
CCTTCTTCTGCACTTCCACA
C2_At5g13
AAGTTTTCCCCATGCCGCTTCTG
200
TATGGGTCCGCCTGCAGTTCCAAC
T
C2_At1g32
AGATTCGGTGTAGAGACTGGAA
410
TGTTAGTGTCTGGAGGGATTGTATTG
GTATC
CSM7F
CGACGATCACCTTGATAACG
CCTTAAATGCAGAGTTTCCAAAG
CSM27
TGTTTGGAGGTGAGGGAAAG
TCCAACTCACCGGAAAAATC
CSM30
CACTGTTCCTGGTTGCTGTG
TTTAGCTTTAGCCCATCTACCG
CSM31
CAACCGATATGCTCAGATGC
CGGGTATGGTCATGTTTTGC
CSM43
ATTTTAACCCCGGGAAAATG
ACCGCTTCTAGGTTTTGCAC
CSM44
CGTCGTTGTAACCCATCATC
TTGCCAAATTCCTTGTGTTC
CSM54
ATGTGCCTCCATTCTGCAAG
TGGGTGGGATGCTGAGTAAG
CSM73
TTCAACATAGCCTGGACCATT
AATGCAGGGTTTGGACTTCA
Number of unique alleles detected in the population
Polymorphism information content for each marker
!"#!

a

b

Al
6
4
3
2
6

PIC
0.18
0.23
0.49
0.03
0.55

Source
SolCAP
SolCAP
SolCAP
SolCAP
SolCAP

5
2

0.69
0.35

SolCAP
SolCAP

7

0.51

SolCAP

2
5
4

0.37
0.62
0.42

SolCAP
NCBI
Minamiyama

2

0.37

NCBI

3

0.10

SolCAP

4
2
3
3
6
4
3
3
4

0.57
0.37
0.50
0.40
0.71
0.55
0.36
0.37
0.56

SolCAP
Hurtado
Hurtado
Hurtado
Hurtado
Hurtado
Hurtado
Hurtado
Hurtado

Table 2.6. Genetic differentiation (pairwise Fst) estimates of SSRs for S. melongena
grouped by continent
b
Fst
N.
a
Category Africa Asia Europe America
Asia 0.00
Europe 0.04* 0.02
N.
America 0.04* 0.00
0.03*
S.
America 0.03 0.11*
0.05
0.06*
a

b

Categories with less than four lines were excluded from analyses and are not shown

Average values for SSRs are presented; * indicates value was outside the 2.5% and
97.5% confidence intervals at 1000 bootstraps
within countries was similar (0.35 – 0.48), and pairwise Fst values suggested low to great
genetic differentiation among countries (Table 2.7, 2.8).
Table 2.7. Genetic diversity estimates for SSRs for S. melongena grouped by continent
and country of origin
b
Diversity estimates
a

b

c

d

Category
AlleleNo
GD
PIC
Africa
2.71
0.51
0.43
Asia
3.10
0.48
0.41
Europe
2.81
0.48
0.41
N.
America
3.10
0.50
0.44
S. America
2.67
0.46
0.39
China
2.18
0.39
0.32
France
2.14
0.38
0.31
Iran
2.23
0.42
0.35
Japan
1.91
0.35
0.28
Macedonia
2.18
0.40
0.33
South
Africa
2.14
0.41
0.34
Spain
2.50
0.42
0.36
Thailand
2.36
0.42
0.36
Turkey
2.36
0.40
0.34
USA
2.77
0.48
0.42
a
Categories with less than four lines were excluded from analyses and are not shown

"#!

!

Table 2.7 (cont’d)
b
Mean values are presented for the average number of alleles (AlleleNo), genetic
diversity (GD) and the polymorphism information content (PIC)
Table 2.8. Genetic differentiation (pairwise Fst) estimates of SSRs for S. melongena
grouped by country
Fst

b

S.
a
Category
China France Iran Japan Mace. Africa Spain Thailand Turkey
France 0.00
Iran 0.00
0.04
Japan 0.01
0.04
0.05
Macedonia 0.06
0.13* 0.09* 0.08
S. Africa 0.04
0.08
0.01
0.07
0.05
Spain 0.06
0.04
0.04 0.13* 0.13* 0.09*
Thailand 0.05
0.04
0.07* 0.15* 0.10*
0.04
0.01
Turkey 0.04
0.09
0.06 0.15* 0.03
0.05
0.09
0.04
USA 0.05
0.10*
0.04 0.17* 0.10*
0.05 0.04*
0.07*
0.05
a
Categories with less than four lines were excluded from analyses and are not shown
b

Average values for SSRs are presented; * indicates value was outside the 2.5% and
97.5% confidence intervals at 1000 bootstraps
Pairwise Fsts for disease resistance to 12889 and OP97 showed little to very great
(0 to 0.52) genetic differentiation between categories (Table 9). No significant genetic
Table 2.9. Genetic differentiation (pairwise Fst) estimates of SSRs for eggplant
germplasm grouped by disease resistance

a

Fst

b

Category
MS
R/MR
S
MS
0.19
0.15
R/MR
0.01
0.52*
S
0.03*
0.00
a
12889 appears below the diagonal and OP97 values are shaded above the diagonal; MS
= moderately susceptible, R/MR = resistant/moderately resistant, S=susceptible
b
Average values for SSRs are presented; * indicates value was outside the 95%
confidence interval at 1000 bootstraps

"$!

!

differentiation was evident between the resistant/moderately resistant (R/MR) category
and the moderately susceptible (MS) or susceptible (S) categories, but significant genetic
differentiation was detected between the MS and the S category for isolate 12889. When
inoculated with isolate OP97, significant differentiation was evident between the S
category and the R/MR categories. There was no significant differentiation between the
R/MR and MS category or the MS and S categories (Table 9). Genetic diversity of fruit
shape categories was moderate to high (0.43-0.52). Pairwise Fst differentiation between
fruit shape categories was low to moderate (0.0-0.1) (Table 2.10). Individuals with an
elongate fruit shape were significantly differentiated from those with a round or oval fruit
shape. Significant differentiation was also detected between round shaped individuals and
semi-elongated individuals (Table 2.10).
Table 2.10. Genetic differentiation (pairwise Fst) estimates of SSRs for S. melongena
germplasm grouped by fruit shape

a

Fst

b

Category
Elongate
Oval
Round
Oval
0.10*
Round
0.06*
0.00
Semi-Elongate
0.04
0.02
0.03*
a
Fruit shape category based on the ratio of mean length: mean length for each line
b

Average values for SSRs are presented; * indicates value was outside the 2.5% and
97.5% confidence interval at 1000 bootstraps.
Population structure of the 99 accessions was estimated using the STRUCTURE
software and the 22 polymorphic SSRs. Accessions were grouped into 4 genetic clusters
(Ln=- 3381.8). S. linnaeanum and S. incanum accessions were placed into genetic cluster
4, while S. melongena individuals were distributed through each of the clusters (Fig 2.3).

"%!

!

Figure 2.3. Population structure grouped by species. Cluster 1 (purple), Cluster 2 (light
yellow), Cluster 3 (sky blue) and Cluster 4 (steel blue).

Seventy-eight individuals could be assigned to a single cluster based on membership,
while the remaining twenty-one individuals could not be assigned and were classified as
admixed.
Pairwise Fsts were significant and ranged from 0.08 to 0.17, indicating 8 – 17%
of the variation was explained by genetic differences between clusters. Cluster 1 had
moderate differentiation from Clusters 2,3 and 4. Cluster 2 had great differentiation from
Cluster 3 and Cluster 4 had moderate differentiation from Clusters 2 and 3 as according
to Hartl and Clark [59]. Population structure was detected when individuals were grouped
by continent of origin, country of origin, species, fruit shape and disease resistance to
12889 and OP97, as some clusters were more frequent than others in each grouping
(Figures 2.4-6).

""!

!

Figure 2.4. Population structure grouped by continent of origin for eggplant germplasm.
Cluster 1 (purple), Cluster 2 (light yellow), Cluster 3 (sky blue) and Cluster 4 (dark blue).
A white space and black tick marks separate subgroups of individuals.

Figure 2.5. Population structure grouped by country of origin for the S. melongena
germplasm. Only countries represented by four or more individuals were included.
Cluster 1 (purple), Cluster 2 (light yellow), Cluster 3 (sky blue) and Cluster 4 (dark blue).
A white space and black tick marks separate subgroups of individuals.

"&!

!

Figure 2.6. Population structure grouped by disease resistance to isolate 12889 (A) and
OP97 (B). Individuals were grouped into a resistant and moderately resistant category
(R/MR), a moderately susceptible category (MS), and a susceptible category (S) based on
their mean disease ratings. Cluster 1 (purple), Cluster 2 (light yellow), Cluster 3 (sky
blue) and Cluster 4 (dark blue). A white space and black tick marks separate subgroups
of individuals.

Cluster 3 individuals were not represented in Asia, and Cluster 4 individuals were not
represented in Africa (Figure 2.4). For both isolates, individuals from Cluster 4 were not
represented in the moderately resistant/resistant categories for either isolate, had low
representation in the moderately susceptible category, and were highly represented in the
susceptible category. Cluster 1 individuals were highly represented in both the R/MR and
S categories, but not the MS for both isolates (Figure 2.5). When grouped by fruit shape
(round, oval, semi-elongate and elongate), Cluster 1 was under represented in the oval
"'!

!

and elongate fruit shape categories. Cluster 4 and 2 both had low representation in the
round category (Figure 2.7). Cluster 3 was not represented in round or elongated
individuals and had minor representation in the oval shape category.
Figure 2.7. Population structure grouped by S. melongena fruit shape. Cluster 1 (purple),
Cluster 2 (light yellow), Cluster 3 (sky blue) and Cluster 4 (dark blue). A white space
and black tick marks separate subgroups of individuals.

DISCUSSION
This study investigated Phytophthora fruit rot resistance, fruit shape, population
structure and genetic diversity in a worldwide collection of eggplant. The overall estimate
of genetic diversity of the collection was moderate (0.49) in our study, similar to a recent
report on eggplant diversity (52). Bayesian clustering identified four genetic clusters in
the eggplant collection. Most individuals belonged to predominantly one of the four
clusters, while an additional 20% were admixed according to the inferred clustering.
Admixture, an indicator of migration or interbreeding between genetic clusters, was low
in our population. Inferred genetic clusters did not directly correspond with the
predefined categories of continent, country, fruit shape or Phytophthora fruit rot
resistance, though some clusters did appear more frequently in one category compared to
another.
"(!

!

On eggplant, fruit rot is the most common symptom of P. capsici seen in the field.
Symptoms start as small water-soaked lesions, turning brown and eventually covering the
whole fruit. Advanced symptoms can include complete rotting of the fruit and visible
mycelia on the external surface of the fruit (37). Isolate-specific interactions and partial
fruit rot resistance have been identified in other solanaceous species (tomatoes and
peppers) suggesting a multigenic host response, but no studies have looked at
Phytophthora fruit rot in eggplant (Naegele et. al. unpublished; Granke et al.
unpublished). In our study, the 99 eggplant accessions evaluated demonstrated partial and
isolate-specific resistance to Phytophthora fruit rot. Most lines evaluated were completely
susceptible to both isolates (~90%). Several S. melongena lines displayed isolate-specific
resistance; these individuals were placed into genetic clusters 2 and 3, and were from
Asia, Africa and Europe. These three geographic regions are known centers of eggplant
diversity, and likely harbor additional sources of resistance (52,75,93,121). Only one of
the ninety-nine lines evaluated, a Cluster 3 S. melongena landrace collected in Burkina
Faso in the early 1900s, had complete resistance to both isolates evaluated. This line also
showed high levels of genetic similarity to the wild eggplant relatives, S. incanum and S.
linnaeanum, evaluated. While further evaluation with more isolates is necessary, PI
413784 appears to be a promising source of host resistance to Phytophthora fruit rot in
eggplant.
When categorized by disease resistance (susceptible, moderately susceptible,
moderately resistant and resistant) for each isolate, there was significant genetic
differentiation among eggplant genotypes infected with isolates OP97 or 12889.
Individuals that were resistant and moderately resistant to isolate OP97 were significantly

")!

!

differentiated from individuals that were susceptible. Only susceptible individuals were
significantly differentiated from the moderately susceptible individuals when inoculated
with isolate 12889. These results emphasize the importance of utilizing different P.
capsici isolates when breeding for resistance. The two wild relatives, S. linnaeanum and
S. incanum, showed partial or isolate-specific resistance to the two isolates evaluated in
this study.
When grouped by species, all S. linnaeanum or S. incanum individuals evaluated
were predominantly genetic cluster 3. S. incanum is one of the progenitors of modern
eggplant and has long been part of the eggplant complex (6,34). S. linnaeanum is a more
distantly related relative and has only recently been included as a possible progenitor of
the modern eggplant with limited crossability (46,121). Genetic cluster 3 individuals
were also detected in the S. melongena category, supporting gene movement between S.
melongena and its wild relatives, S. incanum and S. linnaeanum. These S. melongena
individuals may have been misclassified, but are more likely the result of introgression
since the wild species were small fruited and prickly.
Cultivated eggplant, similar to pepper and tomato, is a phenotypically diverse
species with varying levels of genotypic diversity (6,59,70,114). S. melongena fruit
shape, size and color is a byproduct of breeding for regional specific market classes.
Phenotypic evaluation of eggplant fruit shape varied greatly among the S. melongena
accessions evaluated, while the wild species, S. incanum and S. linnaeanum, had no
variation in fruit shape. Maintaining market class variation may be difficult when
incorporating traits like fruit rot resistance, which was most often observed in smallfruited varieties. Studies in peppers and tomatoes have demonstrated that fruit length and

"*!

!

width are correlated, making breeding for one trait, while maintaining the other, difficult
(19,85). Increased fruit length in particular may be difficult to integrate with disease
resistance as it was positively correlated with increased susceptibility. This was similar to
previous work in peppers where fruit shape, but not length was positively correlated with
fruit susceptibility (Naegele et al. unpublished).
Significant differences in fruit shape, length and width were observed among
eggplant lines when grouped by country of origin, representing different market classes,
in this study. Since eggplant has market classes particular to geographic areas, it was
expected that population structure categorized by fruit shapes and country of origin
would correspond with the inferred genetic clusters. Significant differentiation was seen
between S. melongena individuals with elongated fruit shapes and those with round and
oval fruit shapes. Individuals with a round fruit shape were also significantly
differentiated from semi-elongate fruit shape individuals. These results are consistent
with limited breeding among market classes. However, inferred population structure did
not correspond with the fruit categories. While only genetic cluster 3 was not represented
in the round or elongate shape category, all other clusters were represented by at least one
individual in each category.
When grouped by country and continent, significant population structure and
moderate genetic diversity was evident among the categories evaluated. The highest
levels of genetic diversity were seen within the continents of Africa and N. America. The
highest level of genetic diversity for countries was in the USA. The increased genetic
diversity in Africa is likely due to intercrossing with related species, since Africa is the
center of origin for eggplant and wild relatives. The increased genetic diversity in N.

&+!

!

America and the USA may be the result of breeding programs integrating wild relatives
and varieties from around the world. The diversity could also be from the movement of
Asian and European varieties into the US, which may be marketed under different names.
Overall differentiation among countries was similar to the differentiation among
continents, and future core collections should include individuals from areas with high
genetic diversity and genetic differentiation. In particular, genotypes from China were not
significantly differentiated from any other country, while genotypes from Thailand,
Japan, Spain, Macedonia and the U.S. were frequently significantly differentiated from
other countries. Similarly, Asia was not significantly differentiated from populations
from Europe, Africa and N. America, while N. America, Europe and Africa were all
significantly differentiated from each other. Asia, as a center of diversity and
domestication, and in particular genotypes from China may be more akin to the ancestral
population from which these other pools were derived.
Cultivated eggplant, compared to other solanaceous species, is an understudied
crop with worldwide importance. This study provides an overview of the population
structure, genetic diversity and Phytophthora fruit rot resistance of a geographically
diverse set of eggplant. The estimates of genetic diversity and the four genetic clusters
found in this study are likely to be lower than actual genetic diversity and structure of
eggplant due to limited sampling and molecular markers. A previous study using a subset
of SSRs in a smaller collection of eggplant was able to identify more allelic variation at
each locus [3]. While population structure was significant for disease resistance, fruit
shape, continent and country, the genetic clusters did not completely correspond with the
predefined categories in our study. Future studies involving eggplant diversity, disease

&#!

!

resistance and other agronomic traits should aim to include individuals from around the
world for maximum diversity, and will need to consider the effect of population structure
on marker-trait associations.

&$!

!

CHAPTER 3: EVALUATION OF A DIVERSE, WORLDWIDE
COLLECTION OF WILD, CULTIVATED AND LANDRACE PEPPERS
(CAPSICUM ANNUUM L.) FOR RESISTANCE TO PHYTOPHTHORA FRUIT
ROT, GENETIC DIVERSITY AND POPULATION STRUCTURE
ABSTRACT

Pepper is the third most important solanaceous crop in the U.S. and fourth most important
worldwide. One hundred-seventy pepper genotypes representing five continents and 45
countries were evaluated for Phytophthora fruit rot resistance to two isolates of
Phytophthora capsici. Partial resistance and isolate-specific interactions were identified
in the population at both 3 and 5 days post inoculation (dpi). Most lines evaluated were
susceptible or moderately susceptible at 5dpi, and no lines evaluated were completely
resistant to Phytophthora fruit rot. Genetic diversity and population structure were
assessed on a subset of 157 genotypes using 23 polymorphic simple sequence repeats
(SSRs). Genetic diversity was moderate in the population and varied between countries,
continents and disease susceptibility. The program STRUCTURE inferred four genetic
clusters with moderate to very great differentiation among clusters. Significant
population structure was detected when peppers were grouped by predefined categories
of disease resistance, continent and country of origin.

&%!

!

INTRODUCTION
Pepper, a member of the Solanaceae, is an important vegetable commodity grown in
temperate and tropical regions. Worldwide, pepper production is estimated at 25 million
tons each year [USDA ERS]. China is the largest producer of peppers with an
approximate 15.5 million tons produced in 2011 [FAO, 2011]. The U.S. is the sixth
largest producer with an estimated 1 million tons (combined bell and chile peppers)
[FAO, 2011]. Despite its current distribution around the world, pepper is a New World
crop, domesticated and cultivated in South America since 5000-6000 B.C. (86,87).
Pepper, the common term for the genus Capsicum, contains 25 – 30 species, of which,
only five are economically important (48,117). Cultivated pepper consists of the
intercrossing species: Capsicum annuum, C. chinense, C. frutescens, and C. baccatum
(1,42). Another species, C. pubescens, is also a cultivated member of the pepper genus,
but has limited fertility with the other species (77).
Mexico is the center of origin for C. annuum, the primary pepper species grown in
the U.S. (42). The remaining species originated in other parts of Central and South
America, and are predominantly grown in South America and Asia as vegetables, spices
and ornamentals (3,30,42,77). Wild and landrace individuals from centers of origin and
diversity can harbor genetic variability useful for enhancing fruit quality, disease
resistance and other agronomically important traits (7,26,56,108). Variation in closely
related pepper species, in addition to within species market variation, can provide an
additional resource for breeding. Utilizing genetic diversity in closely related cultivated
species and market classes within the same species instead of wild material could reduce
the number of unfavorable characteristics incorporated. Understanding the genetic

&"!

!

diversity and underlying population structure available within populations is essential for
the efficient utilization and testing of germplasm resources (21,23). Unidentified
population structure can confound association mapping results creating false associations
between markers and phenotypes (58,123). These types of false associations could
decrease the success of marker-assisted breeding programs when incorporating traits of
agricultural significance. Algorithms accounting for the population structure can result in
fewer, but stronger, associations between the trait of interest and individual markers when
properly applied (13,125). In peppers, population structure has been evaluated among
cultivated varieties (51), and within geographic regions (77,82,83). In each of these cases,
population structure was detected and could differentiate between locations (geographic
regions) or market type (cultivated varieties) suggesting within species variation is
available (1,3,51,83). Population structure can also provide information on the migration
and interbreeding of species and populations. Population studies between Capsicum
species have shown that admixture is limited, with few ambiguous genotypes
(65,104,115).
Evaluating individuals can identify useful subsets of individuals representing
much of the genetic diversity within a species. Studies on the genetic diversity within
pepper are more common than population structure studies
(3,30,49,54,77,82,83,104,105). Domesticated populations of peppers (landraces and
cultivars) have been evaluated and compared to wild (natural occurring populations)
(3,51,54,77,83,104,115). Cultivated varieties had higher heterozygosity than landraces,
and only slightly lower diversity than wild populations (1,83). The authors suggested that
cultivated varieties maintained high levels of diversity through hybrid generation, while

&&!

!

landraces, which are often maintained through inbreeding, continuously lost
heterozygosity. These results were similar to a study evaluating cultivars and breeding
lines compared to germplasm resources at the INRA (104,105). The authors determined
that there was no significant difference between the genetic diversity within the
germplasm collection and the breeding/cultivated lines, again suggesting that cultivated
varieties may contain high levels of genetic diversity that can be utilized.
Phytophthora capsici Leonian is a devastating oomycete pathogen capable of
causing disease on pepper (fruit and root rot and foliar blight). Sources of resistance to
root rot have been identified in several Capsicum annuum and C. chinense lines (18,63).
Many of the Capsicum annuum lines with resistance exhibit full or partial resistance to
multiple isolates and are small-fruited landraces from Mexico (18,63). Few studies have
looked at Phytophthora fruit rot resistance, and no correlations between resistance to
Phytophthora capsici diseases in peppers have been identified (5,106,109,116). A single
quantitative trait loci (QTL) analysis in a recombinant inbred line (RIL) population
identified minor and major QTL for fruit rot resistance in immature fruit (Naegele et. al.,
unpublished). However, this study was limited to a single source of resistance, Criollo de
Morelos. Other studies on Phytophthora fruit rot have found age-related resistance, but no
correlations with fruit characteristics such as pungency or pericarp thickness. No pepper
cultivars or lines evaluated to date have demonstrated a complete resistance to
Phytophthora fruit rot. Since morphological traits like fruit shape, color, size, and
pungency are not predictive of susceptibility to Phytophthora fruit rot, reliable molecular
markers are needed to incorporate resistance. Identifying individuals with increased fruit
rot resistance and molecular markers associated with that resistance will increase the

&'!

!

probability of identifying and incorporating Phytophthora fruit rot resistance into
commercial cultivars.
The objectives of this study were to evaluate population structure, genetic
diversity and Phytophthora fruit rot resistance in a worldwide collection of C. annuum
and determine if population structure was associated with disease resistance, country or
continent of origin using polymorphic simple sequence repeat (SSR) markers.

MATERIALS AND METHODS
One hundred-seventy genotypes of pepper germplasm (wild, cultivated and
landrace) were requested from the USDA Germplasm Resource Information Network or
commercial seed sources: Johnny’s Seed, Parks Seed and Seedway (www.ars-grin.gov/)
(Table 1). Twenty seeds from each line were sown into 72 cell trays containing a soilless
Table 3.1. Pepper lines used in this study.
Accession
CM334
Early Jalapeño
Grif 9094
Grif 9105
Grif 9109
Grif 972
Jn566
Jn570
Jn571
Jn574
PI 102883
PI 123469
PI 123474
PI 124078
PI 127445
PI 135822
PI 135826
PI 135874

Country
Mexico
USA
Greece
Former Soviet
Mexico
China
USA
USA
USA
USA
China
India
India
India
Afghanistan
Afghanistan
Afghanistan
Pakistan

Continent
N. America
N. America
Europe
Asia
N. America
Asia
N. America
N. America
N. America
N. America
Asia
Asia
Asia
Asia
Asia
Asia
Asia
Asia
&(!

Source
Park's Seed
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
Johnny's Seed
Johnny's Seed
Johnny's Seed
Johnny's Seed
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
!

Table 3.1 (cont’d)
PI 138557
PI 138558
PI 138560
PI 138565
PI 142832
PI 148628
PI 159256
PI 164311
PI 164560
PI 167063
PI 169129
PI 169140
PI 174114
PI 176888
PI 177294
PI 177301
PI 181733
PI 181734
PI 181934
PI 182646
PI 183668
PI 183922
PI 184039
PI 194259
PI 194261
PI 194910
PI 197408
PI 201232
PI 201234
PI 201237
PI 201239
PI 203524
PI 206950
PI 207727
PI 213915
PI 224438
PI 224442
PI 226633
PI 241641
PI 241644
PI 243936
PI 244669
PI 249635
PI 249908
PI 250141

Iran
Iran
Iran
Iran
Iran
Iran
USA
India
Spain
Turkey
Turkey
Turkey
Turkey
Turkey
Turkey
Italy
Lebanon
Lebanon
Syria
Guatemala
Turkey
India
Serbia
Ethiopia
Ethiopia
Ethiopia
Ethiopia
Mexico
Mexico
Mexico
Mexico
Cuba
Turkey
India
Bolivia
Mexico
Nicaragua
Iran
Colombia
Colombia
Turkey
India
India
Portugal
Pakistan

Asia
Asia
Asia
Asia
Asia
Asia
N. America
Asia
Europe
Europe
Europe
Europe
Europe
Europe
Europe
Europe
Asia
Asia
Asia
S. America
Europe
Asia
Europe
Africa
Africa
Africa
Africa
N. America
N. America
N. America
N. America
S. America
Europe
Asia
S. America
N. America
Africa
Asia
S. America
S. America
Europe
Asia
Asia
Europe
Asia
&)!

USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
!

Table 3.1 (cont’d)
PI 257044
PI 257047
PI 257048
PI 257283
PI 260452
PI 262172
PI 262902
PI 263075
PI 263076
PI 263077
PI 263113
PI 263114
PI 264281
PI 264662
PI 267730
PI 269455
PI 269458
PI 273415
PI 281318
PI 281341
PI 281433
PI 298646
PI 298647
PI 302987
PI 322720
PI 339005
PI 339006
PI 339007
PI 339009
PI 339010
PI 339019
PI 339048
PI 339075
PI 339079
PI 339083
PI 339132
PI 342949
PI 357503
PI 357531
PI 368396
PI 369996
PI 371867
PI 379182
PI 385960
PI 390612

Colombia
Colombia
Colombia
Spain
Argentina
Germany
Spain
Former Soviet
Former Soviet
Former Soviet
Former Soviet
Former Soviet
USA
Germany
Cuba
Pakistan
Pakistan
Italy
USA
El Salvador
USA
Spain
Spain
Canada
India
Turkey
Turkey
Turkey
Turkey
Turkey
Turkey
Turkey
Turkey
Turkey
Turkey
Turkey
USA
Serbia
Serbia
Serbia
India
USA
Serbia
Kenya
Peru

S. America
S. America
S. America
Europe
S. America
Europe
Europe
Asia
Asia
Asia
Asia
Asia
N. America
Europe
S. America
Asia
Asia
Europe
N. America
S. America
N. America
Europe
Europe
N. America
Asia
Europe
Europe
Europe
Europe
Europe
Europe
Europe
Europe
Europe
Europe
Europe
N. America
Europe
Europe
Europe
Asia
N. America
Europe
Africa
S. America
&*!

USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
!

Table 3.1 (cont’d)
PI 409141
PI 410407
PI 427290
PI 432802
PI 432818
PI 438565
PI 438624
PI 438633
PI 441628
PI 511879
PI 511882
PI 511884
PI 511886
PI 550700
PI 555649
PI 566808
PI 566811
PI 585246
PI 593493
PI 593495
PI 593511
PI 593561
PI 593564
PI 593572
PI 593573
PI 593920
PI 593929
PI 593933
PI 595906
PI 600934
PI 601110
PI 631126
PI 631131
PI 631140
PI 631143
PI 631147
PI 639641
PI 640448
PI 640460
PI 640461
PI 640480
PI 640516
PI 640532
PI 640560
PI 640579

South Africa
Brazil
USA
China
China
Guatemala
Mexico
Mexico
Brazil
Mexico
Mexico
Mexico
Mexico
USA
Sudan
Mexico
Mexico
Ecuador
Mexico
Mexico
Mexico
USA
Mexico
Brazil
Brazil
Ecuador
Venezuela
Ecuador
Venezuela
USA
USA
China
Yemen
Guatemala
Guatemala
India
Poland
Taiwan
China
China
France
Taiwan
Mexico
Netherlands
Egypt

Africa
S. America
N. America
Asia
Asia
N. America
N. America
N. America
S. America
N. America
N. America
N. America
N. America
N. America
Africa
N. America
N. America
S. America
N. America
N. America
N. America
N. America
N. America
S. America
S. America
S. America
S. America
S. America
S. America
N. America
N. America
Asia
Asia
N. America
N. America
Asia
Europe
Asia
Asia
Asia
Europe
Asia
N. America
Europe
Africa
'+!

USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
!

Table 3.1 (cont’d)
PI 640581
PI 640582
PI 640588
PI 640641
PI 640659
PI 640663
PI 640670
PI 640671
PI 640676
PI 640682
PI 640744
PI 640791
PI 640803
PI 640809
PI 640815
PI 640833
PI 645520
PI 653650

Nigeria
Nigeria
USA
Indonesia
Thailand
Taiwan
India
Sri Lanka
Kenya
Tanzania
Japan
Egypt
Philippines
Denmark
Zambia
USA
Italy
Bangladesh

Africa
Africa
N. America
Asia
Asia
Asia
Asia
Asia
Africa
Africa
Asia
Africa
Asia
Europe
Africa
N. America
Europe
Asia

USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN
USDA GRIN

peat mix (Suremix Michigan Grower Products, Inc Galesburg, MI) in a polyethylene
greenhouse at the Michigan State University Horticulture Research Farm (East Lansing,
MI.) Germinated seedlings were transferred to 4” black plastic pots containing the same
soilless peat mix and grown to maturity. Immature fruit were detached, and bulked for
each line. Fruit were taken to the laboratory for inoculation and subsequent evaluation.
Two isolates were selected from the long-term collection of Dr. Mary K.
Hausbeck (Michigan State University). Isolates were characterized by host, mefenoxam
sensitivity (insensitive=I, sensitive=S) and mating type (A1 or A2). Isolate 12889
(pepper, I, A1) and isolate OP97 (cucumber, S, A1) were maintained on unclarified V8
agar during the experiment. Prior to inoculations, isolates were activated by inoculating
and recovering each isolate from a single pepper fruit.
Peppers were surface disinfested in 10% bleach for 5 minutes, rinsed with
distilled water, and dried prior to inoculation. For inoculations, a single 6mm plug of V8
'#!

!

agar from the actively growing isolate was placed, mycelium side down, onto the surface
of the fruit. Plugs were covered with a sterile microcentrifuge tube cap and affixed into
place with petroleum jelly. Peppers were placed into a humidity chamber consisting of a
clear, covered plastic box with a ring of moistened paper towel around the outside edge,
and placed on a light bench under constant light at room temperature (25 °C). Peppers
were blocked by isolate and completely randomized within the boxes. Two control
peppers were inoculated with a sterile plug of V8 agar for each line. Five pepper fruit
from each accession per isolate were evaluated and the experiment was performed three
times for a total of 15 peppers per isolate, per line. Pepper landrace CM334 was used as
the resistant control and Johnny’s Breeding line JN571 was evaluated as the susceptible
control. Due to poor fruit set only two experimental replicates were performed on lines
Grif 972, JN570, 262902, 339083, 511886, 593572, 631131, 631140, 640803, 182646,
194261, and197408 when inoculated with isolate OP97 and lines 194261, 182646,
164560, 640641 631131, 631126, 593572, 593511, 511882, 438633, 566811, 123469,
273415, JN570, and 224442 with isolate 12889.
Peppers were evaluated at 3 and 5 days post inoculation (dpi). Lesion area in cm2
(maximum length by maximum width) was measured using a hand caliper. For some
accessions the visible lesion area was not solidly filled (e.g. multiple spots of diseased
tissue) and a coverage score was determined. If symptomatic tissue covered less than
25% of the lesion area measured, the coverage score=0.25, 25% to <50% the coverage
score was 0.5, if symptoms covered 50% to <75% the score =0.75, if the visible lesion
coverage was >75% the coverage score =1. For recovery of the isolates, isolations were
performed on approximately 10% of the symptomatic fruit. Three pieces of fruit were

'$!

!

plated onto V8 agar amended with ampicillin, rifampicin and benomyl (2mL, 2mL, and
0.05g, respectively per liter). Cultures of P. capsici were identified by morphological
characteristics according to Waterhouse et al (120). Mefenoxam sensitivity was
confirmed by transferring isolates to V8 plates amended with 100ppm mefenoxam and
determining growth.
Control fruits (inoculated with sterile V8 plug) were removed prior to analysis to
avoid violating variance assumptions. Experimental replicates were combined for
statistical analysis. Final lesion area was calculated by multiplying the lesion area by the
coverage score. Final lesion score for 3dpi and 5dpi relative to line, country of origin and
host of origin, was analyzed by ANOVA using the PROC MIXED function of SAS v9.3.
Line, isolate and interaction means were separated using LSD at P=0.05, when
significant. When analyzing unequal sample sizes, degrees of freedom were accounted
using the Kenward Rogers option implemented in SAS. Countries represented by less
than four lines were removed from analyses. Data was log transformed for 3dpi values
2

when analyzing by line and transformed 1/x where x indicates the line value at 3 and
5dpi when analyzed by countries and continents to normalize the data. The percentage of
diseased fruit was calculated by combining all replicates for a single line and isolate (total
diseased out of 15 total peppers). A diseased fruit was considered to be fruit with a
visible lesion, regardless of lesion size.
Genomic DNA was extracted from young green leaves of peppers using the
Nucleo Spin II DNA extraction kit (Machery-Nagel Germany, CAT#740770) according
to the manufacturer’s instructions. DNA was normalized to 10 ng/ul using the NanDrop

'%!

!

ND 1000 spectrophotometer and NanoDrop 2.4.7c software (NanoDrop Technologies
Inc., Wilmington, DE).
One hundred ninety-two primers from previously published SSR markers (76),
solgenomics.org or designed (Primer 3 http://primer3.sourceforge.net/) from putative
Solanaceae defense-related genes (NCBI ncbi.nlm.nih.gov) were tested against a subset
of the pepper collection to identify polymorphic markers. Reactions were performed in
15 ul total volume and contained 1 ul DNA, and 0.15 ul GoTaq (Promega Corporation
Madison, Wisconsin), 0.9 ul 25uM MgCl2, 0.3 ul dNTPs, and 0.6 ul each of forward and
reverse primers (Integrated DNA Technologies, Inc.), with 8.45 ul ddH2O. PCR reactions
were performed in a programmable thermal cycler (Eppendorf, Westbury, NY) using the
program: initial denaturation, 94 C (3 min) followed by 35 cycles at 94 C (30s), 60 C
(30s) and 72 C (1 min), with a final extension step of 10 min at 72 C. PCR products
wereanalyzed by electrophoresis in 4% (wt/vol) agarose gel in 1x Tris-borate-EDTA
buffer, stained with ethidium bromide (5 ug/ml) for visualization and compared to a 100bp ladder (Invitrogen Life Technologies Burlington, ON Canada) to determine amplicon
sizes. SSR markers identified as polymorphic in the population were used for genetic
diversity, and population structure analyses (Table 3.2).

'"!

!

Table 3.2. Simple sequence repeat (SSR) markers tested against the pepper genotypes and their respective genetic diversity
and polymorphism information content (PIC) within the population.
SSR
E492334

Primer sequence
Source
GCTGGTTGTGGTTGTACGAG
TGCTCACATATCAATAGATTCAGC
SOLCAP
AAGCCTCCTTGACAAATGCATAT AGATATAGCTACAGTGGCAGCTTCA
U221402
AG
TC
SOLCAP
T0633
GATGGGCTATGCTTGCTGTT
ACATCCCCAATGTTGTTGTG
SOLCAP
CAAACTATTTCAGATTTACACTT ACCGTTCAAGTTGGCTCTTCACAAC
C2_At2g30100
AAATG
AG
SOLCAP
CA516044
ATCTTCTTCTCATTTCTCCCTTC
TGCTCAGCATTAACGACGTC
SOLCAP
GGAAGATCCCTTGAATGAGTATG GGCTGAAAATGTCTGATGGAACTG
asu5
TCTC
G
SOLCAP
AAGAACATGAGGAACTTTAACC
GPMS159
ATG
TTCACCCTTCTCCGACTCC
SOLCAP
GP20117
TGACAGCTACCGAAAATGA
CCTCTAATGCTGACGTGAA
SOLCAP
CP10023
CACCATGTAGCATCTGGG
GATGGATGGATCGACAGA
SOLCAP
GP1127
CACCACCAGTCACAAAGTTAC
CCCTTCAAATACATCCCATGC
SOLCAP
CA515275
CTCTGCCCTCCTCAACCC
AAAATATGGTCGGAGATCCG
SOLCAP
CP10023
CACCATGTAGCATCTGGG
GATGGATGGATCGACAGA
SOLCAP
GP20087
CCCTCTCCTCAATTCACA
CCTTTACCCCTAAATTTGAT
SOLCAP
GQ386945
GCTGCTATGCCCCCAAGGAT
TTTCTAGACAAGGCAGCTCACCAAT
NCBI
AF348141
CCTTACGGGGAAAACCTAGC
CCATACGGACGTTGTCCTCT
NCBI
EF645679
GCGCGAGAGACTACAAATCC
CACTCCTTCGTATCCCTCCA
NCBI
EF100893
GTTTGGTCTTGTGGGGTCAC
GGCTTTTCTCCACCATTCAC
NCBI
GU295217
TTTCGGATTGCCCTATGCTTGTT
AAATTTGTGAGGGCTGTTAGGT
NCBI
ATTCGGGGTGTGATGAGGTGGA AAAAACAAACATAGGGCAAGACGA
GU116570
G
A
NCBI
HPMSHSMAD TGCTTTCAAAACAATTTGCATGG GCGTCTAATGCAAAACACACATTAC Minamiyama
"#!

a

GD
0.51

PIC
0.44

0.57
0.26

0.52
0.24

0.24
0.63

0.22
0.58

0.33

0.31

0.38
0.49
0.48
0.54
0.21
0.55
0.28
0.26
0.64
0.62
0.29
0.14

0.32
0.45
0.44
0.46
0.20
0.52
0.26
0.25
0.57
0.54
0.27
0.13

0.13
0.70

0.12
0.65

!

Table 3.2 (cont’d)
CAMS319
TCACCTTCCACAGCATCAAG
CAMS839
GCAAGCACATCATGCTGAAT
TATGGGTCCGCCTGCAGTTCCAA
C2_At5g13200
C
a
Genetic diversity for each marker
b

CAAACGCAAACACCAATCAG
CGAGCGCATTATTGAAGTGA

Minamiyama
Minamiyama

0.50
0.61

0.42
0.57

AAGTTTTCCCCATGCCGCTTCTGT

SOLCAP

0.75

0.71

Polymorphism information content for each marker

""!

!

Genetic diversity was estimated using Powermarker v3.25 (71) and significance at
each locus was determined with 1000 permutations using the Exact test; overall genetic
diversity was estimated using the Mantel test as implemented in Powermarker.
Population structure of the germplasm was analyzed using STRUCTURE v2.3.4
(92). Following preliminary analyses, burnin length, MCMC chain replication and
lambda were selected to be 200,000, 500,000 and 0.49, respectively. Population number
(k) was determined empirically by comparing posterior distribution likelihoods
independently among 3 independent runs of K=1 to 20 as described by Evanno et al. (27).
Data included 23 polymorphic SSRs and were analyzed using the admixture model and
correlated allele frequencies without previous population information (28,92). Fst
significance between populations was determined using 95% confidence intervals based
on 1000 bootstrap replicates as implemented in Powermarker.
Visualization of the resulting Q (proportion of membership) of each individual
into predefined categories (country, continent, and disease susceptibility) was generated
using the Population Sorting Tool (PST) in R (99), J.J. Morrice unpublished.) Individuals
with Q!0.6 membership in a single subpopulation were labeled as such. Individuals with
Q<0.6 membership in a single subpopulation were considered admixed. Significant
population structure by continent, country and Phytophthora fruit rot resistance was
estimated using the population differentiation test implemented in Powermarker.
Significance at each locus and overall was determined using 1000 permutations.
Countries represented by less than four individuals were excluded from analyses.
Significance of pairwise Fst differentiation was based on 2.5 and 97.5% confidence
intervals calculated from 1000 bootstrap replications. At 3dpi, fruit were grouped into

"#!

!

2

2

resistant (lesion area 0<2 cm ), moderately resistant 2<5cm , moderately susceptible
2

2

(5<10cm ) and susceptible (10 cm and greater) categories. At 5dpi, fruit categories were
2

2

resistant (0<5 cm ), moderately resistant (5<10 cm ), moderately susceptible (10 <20
2

2

cm ) and susceptible (20 cm and greater).

RESULTS
Significant differences were identified among pepper genotypes at both 3 and
5dpi. At 3dpi, isolate was not significant (P=0.319) and line was highly significant
(P<0.001). Line-by-isolate interactions were also significant (P=0.0001) at 3dpi.
Breeding line JN571 was the most susceptible with an average lesion area of 27 and 30
2

cm for isolates 12889 and OP97, respectively. When inoculated with isolate OP97 line
2

PI 640516 had the smallest average lesion area of 0 cm . When inoculated with 12889,
2

line 640803 had the lowest lesion area (0 cm ). Most lines, 111 and 121 for isolates
12889 and OP97, respectively, were moderately resistant or resistant to Phytophthora
capsici at 3dpi.
Significant line-by-isolate interactions (P=0.0008) were identified at 5dpi. Line
was highly significant (P<0.0001) and isolate was not significant (P=0.302). Most
genotypes evaluated were susceptible to both isolates: 74 lines for isolate OP97 and 103
for 12889 (Table 3). No accession was completely resistant to both isolates of P. capsici.
2

JN571 had an average lesion area of 50 cm , indicating the fruit were completely covered

"$!

!

Table 3.3. Pepper fruit disease susceptible at 3 days post inoculation (dpi) and 5dpi when
inoculated with isolates OP97 and 12889.
OP97
12889
a
a
a
a
Day 3
Day 5
Day 3
Day 5
PI
% Incidence
% Incidence
CM334
1.06
17.32
0.53
1.37
14.42
0.93
Early
Jalapeño
2.69
43.33
0.87
2.80
28.63
1.00
Grif 9094
9.71
48.50
1.00
9.98
46.23
1.00
Grif 9105
8.47
46.97
1.00
6.35
44.12
1.00
Grif 9109
1.56
14.60
0.47
1.37
16.68
0.47
Grif 972
5.10
27.80
0.70
Jn566
3.72
36.71
0.80
4.80
35.07
0.87
Jn570
17.44
49.91
1.00
12.65
50.81
1.00
Jn571
28.24
50.00
1.00
30.49
53.33
1.00
Jn574
6.77
35.51
0.87
9.76
46.92
1.00
PI 102883
3.06
25.26
0.87
PI 123469
1.75
30.00
0.60
1.58
34.26
0.80
PI 123474 10.98
34.87
0.87
6.74
37.39
0.80
PI 124078
1.40
27.71
0.60
1.03
20.77
0.47
PI 127445
2.38
26.67
0.53
4.20
38.09
0.80
PI 135822
2.89
27.39
0.60
2.08
23.50
0.60
PI 135826
3.15
32.02
0.80
2.63
27.45
0.67
PI 135874
1.87
21.09
0.53
1.15
20.32
0.47
PI 138557
4.58
34.34
0.73
2.51
25.32
0.67
PI 138558
2.49
31.89
0.73
4.43
34.38
0.80
PI 138560
6.59
34.87
0.80
2.35
29.05
0.73
PI 138565
1.58
17.31
0.40
1.76
27.43
0.79
PI 142832
3.26
36.52
0.86
3.26
34.25
0.85
PI 148628
2.13
36.68
0.80
5.18
30.00
0.60
PI 159256
4.49
32.56
0.73
4.25
36.55
0.87
PI 164311
4.79
37.30
0.87
10.38
43.33
0.87
PI 164560
1.26
26.38
0.73
2.95
30.03
0.70
PI 167063
8.61
48.90
1.00
6.01
46.76
1.00
PI 169129
7.94
39.32
0.93
9.07
45.10
1.00
PI 169140
1.57
18.28
0.53
3.36
33.70
0.87
PI 174114
1.25
22.00
0.47
2.19
33.34
0.80
PI 176888
5.38
30.43
0.73
8.89
37.33
0.80
PI 177294
5.78
33.16
0.67
3.42
30.80
0.80
PI 177301 11.94
46.21
1.00
5.87
35.12
0.73
PI 181733
3.00
33.55
0.73
4.62
38.46
0.87
PI 181734
2.41
27.54
0.80
9.23
46.67
0.93
PI 181934
5.46
41.48
0.93
PI 182646
1.33
31.02
0.70
2.47
35.81
0.70
PI 183668
2.06
21.53
0.53
2.54
33.67
0.73
PI 183922
3.22
37.40
0.80
3.40
36.67
0.80
PI 184039
5.76
38.75
0.93
5.90
44.19
0.93
"%!

!

Table 3.3 (cont’d)
PI 194259
1.41
PI 194261
1.66
PI 194910
1.52
PI 197408
1.21
PI 201232
1.48
PI 201234
1.33
PI 201237
1.28
PI 201239
4.39
PI 203524
1.75
PI 206950
4.40
PI 207727
PI 213915
1.36
PI 224438
1.06
PI 224442
PI 226633
4.64
PI 241641
1.27
PI 241644
7.60
PI 243936
6.48
PI 244669
1.61
PI 249635
1.75
PI 249908
6.66
PI 250141
2.20
PI 257044
1.96
PI 257047
1.52
PI 257048
4.21
PI 257283
1.77
PI 260452
2.24
PI 262172
5.51
PI 262902
3.62
PI 263075
4.21
PI 263076
1.18
PI 263077
6.17
PI 263113
1.74
PI 263114
2.07
PI 264281
1.29
PI 264662
6.06
PI 267730
3.71
PI 269455
1.09
PI 269458
1.75
PI 273415
2.00
PI 281318
1.53
PI 281341
PI 281433
1.52
PI 298646
1.58
PI 298647
4.34

13.33
16.82
19.76
23.22
15.54
30.62
9.59
27.31
22.46
39.17
10.06
13.72
37.49
23.84
36.67
32.21
26.67
27.83
46.67
13.33
25.78
19.03
31.70
25.52
30.64
43.10
25.54
40.12
39.27
46.67
46.67
35.70
11.81
42.70
20.00
10.06
28.39
21.86
23.97
30.50
18.33
47.48

0.27
0.50
0.53
0.70
1.00
0.73
0.60
0.67
0.60
0.87
0.33
0.60
0.80
0.53
0.73
0.87
0.53
0.60
0.93
0.27
0.67
0.53
0.67
0.73
0.67
1.00
0.60
0.93
0.93
0.93
0.93
0.87
0.40
0.93
0.40
0.27
0.60
0.80
0.67
0.67
0.53
1.00

3.71
6.18
5.96
1.51
2.23
1.06
1.75
1.02
4.60
3.05
4.23
1.54
6.32
7.39
1.11
6.22
2.21
2.55
8.68
4.08
1.32
1.83
2.29
3.14
3.26
1.81
5.63
1.63
2.54
1.73
4.51
7.23
1.50
1.31
6.25
2.20
4.12
1.65
1.64
6.41
2.48
1.77
1.91
#&!

33.33
35.81
36.81
13.02
30.90
4.07
24.14
14.71
40.06
33.33
26.67
28.64
40.81
46.93
14.08
32.20
28.89
35.62
42.92
40.96
14.06
30.95
24.85
35.44
36.91
30.73
37.10
24.58
32.02
28.01
36.65
50.00
17.26
16.13
44.75
26.67
28.83
16.00
28.10
45.92
30.00
23.60
30.52

0.67
0.70
0.80
0.80
0.87
0.60
0.80
0.47
0.93
0.67
0.60
0.64
0.80
1.00
0.73
0.53
0.80
0.87
0.86
1.00
0.33
0.67
0.80
0.87
0.93
0.67
0.93
0.73
0.60
0.80
1.00
0.73
0.60
1.00
0.53
0.67
0.50
0.73
1.00
0.60
0.67
0.93
0.87
!

Table 3.3 (cont’d)
PI 302987
4.62
PI 322720
1.39
PI 339005
5.01
PI 339006 10.88
PI 339007
2.46
PI 339009
4.33
PI 339010
2.05
PI 339019
6.60
PI 339048
2.16
PI 339075 11.44
PI 339079 10.59
PI 339083
6.98
PI 339132
3.86
PI 342949
6.14
PI 357503 24.80
PI 357531
3.73
PI 368396 16.78
PI 369996
3.43
PI 371867
2.02
PI 379182
3.47
PI 385960
7.38
PI 390612
8.83
PI 409141
1.04
PI 410407
4.89
PI 427290
1.13
PI 432802
6.58
PI 438565
1.08
PI 438624 17.87
PI 438633 12.58
PI 441628
1.91
PI 511879
1.75
PI 511882
1.86
PI 511884
1.58
PI 511886
2.33
PI 550700
3.34
PI 555649
1.96
PI 566808
7.21
PI 566811
1.08
PI 585246
2.41
PI 593493
1.00
PI 593495
1.91
PI 593511
1.74
PI 593561
2.59
PI 593564
6.68
PI 593572
2.17

40.72
7.09
26.67
48.50
30.49
36.19
27.42
30.00
25.70
46.87
44.90
40.20
31.13
34.77
45.61
42.85
45.59
46.14
25.80
30.23
40.80
40.00
20.00
45.21
7.57
47.68
26.67
40.00
45.57
18.65
23.65
14.05
30.75
27.66
39.76
24.34
35.86
0.66
37.17
16.67
30.02
20.01
39.85
40.00
20.99

0.87
0.20
0.53
1.00
0.73
0.80
0.60
0.60
0.60
1.00
0.93
0.80
0.67
0.87
1.00
1.00
0.93
0.93
0.80
0.67
0.93
0.80
0.40
1.00
0.27
1.00
0.47
0.80
0.93
0.73
0.67
0.87
0.87
0.56
1.00
0.80
0.73
0.69
0.87
0.67
0.80
0.53
0.87
0.80
0.50

4.36
1.41
17.70
17.68
10.17
10.13
10.33
10.50
3.65
14.67
9.01
3.87
4.26
26.41
8.99
16.76
2.29
3.05
8.89
10.31
5.63
1.42
19.38
1.64
8.08
13.92
20.72
2.05
1.41
5.21
1.07
3.56
7.51
3.12
17.12
1.06
3.54
1.69
1.10
1.31
3.75
2.85
5.40
#'!

42.52
24.92
45.51
46.67
50.00
41.48
45.57
47.06
31.53
50.31
40.13
35.50
38.66
46.94
44.91
50.10
29.63
28.40
48.05
47.84
31.91
30.05
47.26
26.80
42.32
46.67
48.86
30.53
13.62
36.05
11.28
20.81
45.95
37.27
47.21
3.81
36.68
23.33
23.53
20.82
40.19
30.00
34.33

0.60
0.93
0.93
1.00
0.93
0.93
1.00
0.67
1.00
0.80
0.73
0.93
0.93
1.00
1.00
0.93
1.00
1.00
1.00
0.67
0.73
0.93
0.73
0.93
0.93
1.00
0.73
0.47
1.00
0.60
0.44
1.00
0.87
1.00
0.60
0.87
0.47
1.00
0.50
0.93
0.60
0.67
0.53
!

Table 3.3 (cont’d)
PI 593573
1.78
20.92
PI 593920
1.77
25.03
PI 593929
3.26
39.10
PI 593933
6.15
33.33
PI 595906
4.82
30.00
PI 600934
3.18
31.67
PI 601110
9.04
43.34
PI 631126
2.80
39.32
PI 631131
5.27
34.55
PI 631140
1.10
32.63
PI 631143
5.38
35.70
PI 631147
2.78
33.35
PI 639641
7.68
37.80
PI 640448
1.59
29.76
PI 640460
4.65
36.00
PI 640461
2.34
28.56
PI 640480
3.59
37.21
PI 640516
1.00
30.76
PI 640532
1.75
18.21
PI 640560
3.39
36.03
PI 640579
6.97
34.64
PI 640581
1.36
21.49
PI 640582
1.01
6.67
PI 640588
1.66
34.03
PI 640641
1.04
6.86
PI 640659
1.12
20.05
PI 640663
1.93
20.73
PI 640670
1.69
26.67
PI 640671
6.58
37.32
PI 640676
1.00
24.63
PI 640682
1.72
20.00
PI 640744
1.93
37.86
PI 640791
9.43
41.60
PI 640803
1.18
12.00
PI 640809
3.90
37.19
PI 640815
2.02
16.67
PI 640833
1.23
8.26
PI 645520
7.44
45.24
PI 653650
1.10
14.27
a
2
Mean lesion area (cm )

0.47
0.50
0.87
0.67
0.60
0.93
0.93
0.93
0.80
0.67
0.71
0.73
0.93
1.00
0.87
0.57
0.87
0.69
0.73
0.80
0.93
0.50
0.13
0.87
0.33
0.60
0.93
0.53
0.80
0.79
0.40
0.93
0.87
0.70
1.00
0.33
0.85
1.00
0.29

2.34
1.37
1.97
2.79
2.21
2.18
24.41
2.37
3.24
1.31
1.71
1.43
18.75
2.43
3.44
5.30
2.92
2.35
2.09
3.08
3.93
1.07
1.53
1.93
2.03
1.47
1.63
2.54
9.41
1.16
1.94
1.56
13.68
0.97
2.90
2.35
1.25
12.12
1.30

18.08
20.00
32.38
35.77
13.40
23.45
47.26
20.23
28.87
36.67
33.33
17.68
50.82
20.67
35.65
35.82
41.53
25.12
19.35
35.16
40.60
3.78
26.72
33.93
26.40
18.27
26.23
28.69
37.13
24.83
33.44
26.31
44.10
5.68
30.00
30.00
0.42
48.79
16.68

0.40
0.93
0.73
0.33
0.87
1.00
1.00
0.70
0.73
0.67
0.47
1.00
0.87
0.93
0.80
0.93
0.50
0.86
1.00
1.00
0.57
0.60
0.73
0.60
0.80
0.93
0.57
0.93
0.79
0.73
0.93
1.00
0.30
0.67
0.67
0.93
1.00
0.40
0.93

2

by the lesion. CM334, the resistant control, had an average lesion area of 14 cm and 17
2

cm when inoculated with isolates 12889 and OP97, respectively. One line, PI 566811
#(!

!

2

had an average lesion area of 1 cm and was significantly more resistant than CM334
when inoculated with OP97. This same line had a lesion area of 3.8 cm2 when inoculated
with isolate 12889.
When analyzed by country and continent of origin significant differences were
evident at 3 and 5dpi (Table 3.4, 3.5). Country was highly significant (P<0.0001), isolate
was
Table 3.4. Capsicum spp. Phytophthora fruit rot resistance among countries of origin

a

Lesion area

b

Category
3dpi
5dpi
Pakistan
5.66 A
19.44 A
Ethiopia
9.21 AB
25.56 BC
Mexico
7.58 B
23.64 B
Colombia
9.71 B
27.41 BCD
Guatemala
5.89 A
33.01 BCDE
India
9.47 BC
31.28 CDE
Brazil
10.13 CD
29.55 BCDE
Iran
10.78 D
32.60 DEF
Spain
4.88 BC
29.44 EFG
USA
8.81 E
33.16 FGH
Turkey
13.69 E
36.78 FGH
Soviet
7.61 E
38.62 HI
China
11.23 E
34.55 GHI
Serbia
18.82 F
43.72 I
a
Categories with less than four individuals representing a country were not included in
analyses
b
2
Mean lesion area (cm ) for both isolates combined at 3 days post inoculation (3dpi) and
5 days post inoculation (5dpi); different letters within a time period indicate significant
differences among transformed values using LSD at P=0.05 for both isolates

#)!

!

Table 3.5. Capsicum spp. Phytophthora fruit rot resistance among continents of origin
Lesion area
a

3dpi

12889
OP97
Category
Africa
10.29 BCD 5.89 A
Asia
9.28 C
7.77 BC
Europe
14.28 E
11.50 D
N. America 8.26 C
7.84 BC
S. America 9.79 BC
9.49 B

a

b

5dpi
12889
33.10
30.50
39.88
29.34
28.84

BC
BC
D
B
B

OP97
23.31
30.71
35.67
28.39
28.17

A
BC
C
B
B

Categories with less than four individuals representing a country were not included in
analyses
b
2
Mean lesion area (cm ) for both isolates combined at 3 days post inoculation (3dpi) and
5 days post inoculation (5dpi); different letters within a time period indicate significant
differences among transformed values using LSD at P=0.05 for both isolates
also significant (P<0.0001 and P=0.007), and the interaction between country and isolate
was not significant (P=0.3448 and P=0.0581) at 3 and 5dpi. Fruit from Pakistan were the
least susceptible, while fruit from Serbia were the most susceptible at both 3 and 5dpi.
When analyzed by continent, significant interactions (P=0.0369 and P=0.0269) were
detected between continent and isolate at 3dpi and 5dpi. Isolate OP97 was significantly
more virulent than isolate 12889 at both 3 and 5dpi. At 3dpi, fruit from Africa and S.
America were the least susceptible when inoculated with OP97 and 12889, respectively.
Fruit from Europe were the most susceptible when inoculated with 12889 or OP97 3dpi.
At 5dpi, fruit from Africa and S. America were the least susceptible and fruit from
Europe were the most susceptible when inoculated with OP97 and 12889, respectively
(Table 3.5).
The percentage of diseased fruit varied greatly (13-100% incidence) between lines
and isolates at 5dpi (Table 3.3). Most lines evaluated had incidence greater than 50%,
#*!

!

150 and 148 for isolates 12889 and OP97, respectively. When inoculated with isolate
12889, line PI 640803 had the lowest incidence with 30% diseased fruits. CM334 had a
disease incidence of 93%. Line JN571 had 100% disease incidence as did 32 other lines.
When inoculated with isolate OP97, line PI 640582 had the lowest disease incidence with
13% of fruits infected. CM334 had a disease incidence of 53%. JN571 had 100% disease
incidence along with 18 other lines.
The 192 SSRs evaluated yielded 23 polymorphic markers that were used for
characterizing and evaluating population structure of the pepper collection. Population
structure for 155 of the 170 genotypes was estimated using the Bayesian analysis
software, STRUCTURE (92). Pepper lines were grouped into 4 genetic clusters (Ln=5058). The two C. frutescens and C. chinense accessions were placed into genetic cluster
3, while C. annuum individuals were distributed through each of clusters (Figure 3.1).
One hundred forty-two individuals could be assigned to a single cluster based on
membership. The remaining fifteen individuals could not be assigned to a single cluster
and were classified as admixed. For the STRUCTURE inferred clusters, mean Fsts varied
from 0.12 to 0.45. Cluster 1 had moderate, very great, and little differentiation from
clusters 2, 3, and 4, respectively. Cluster 2 had moderate and very great differentiation
from cluster 3 and cluster 4, respectively. Cluster 3 had very great differentiation from
clusters 1, 2 and 4 as according to Hartl and Clark (45).
When individual genotypes were grouped by continent and country of origin,
species or diseases resistance, population structure was detected. Significant differences
were detected among individual markers and the predefined categories. No clusters were
perfectly correlated with predefined categories, but some clusters were more frequently

#+!

!

found in some categories. Capsicum frutescens and C. chinense individuals were only
represented by cluster 3 (Figure 3.1).

Figure 3.1. Population structure grouped by species. Cluster 1 (purple), Cluster 2 (light
yellow), Cluster 3 (sky blue), Cluster 4 (steel blue).

Cluster 3 individuals were only represented in S. America and Africa, and cluster 1
individuals less prevalent in Africa and S. America (Figure 2).
Figure 3.2. Population structure grouped by continent of origin for pepper germplasm.
Cluster 1 (purple), Cluster 2 (light yellow), Cluster 3 (sky blue), Cluster 4 (steel blue). A
white space and black tick marks separate subgroups of individuals.

Cluster 2 individuals less prevalent in Africa, but constituted a higher proportion of
individuals from Europe, S. America and Asia. Cluster 4 individuals were a low
#"!

!

proportion of individuals from Europe and S. America. Among countries, variation in
cluster representation was more evident than among continents (Figure 3.3).
Figure 3.3. Population structure grouped by country of origin for the C. annuum
germplasm. Only countries represented by four or more individuals were included.
Cluster 1 (purple), Cluster 2 (light yellow), Cluster 3 (sky blue), Cluster 4 (steel blue). A
white space and black tick marks separate subgroups of individuals.

Individuals in cluster 1 were not found in China, Colombia, or Spain. Individuals from
cluster 2 were not found in Brazil or China. Cluster 3 individuals were not found in any
of the countries represented by at least four lines. Cluster 4 was only represented in
individuals from Brazil, China, India, Mexico Turkey and the USA. At 3dpi, each cluster
was represented in the resistant category for isolates OP97 and 12889. When grouped by
disease resistance for both isolates at 5dpi, individuals from clusters 3 and 4 were not
represented in the moderately resistant/resistant categories and cluster 2 had a low
prevalence in the resistant/moderately resistant categories for isolate 12889 (Figures 3.4,
3.5).

##!

!

Figure 3.4. Population structure grouped by Phytophthora fruit rot resistance to isolate
OP97 at A) 3 days post inoculation and B) 5 days post inoculation. Individuals were
grouped into a resistant and moderately resistant category (R/MR), a moderately
susceptible category (MS), and a susceptible category (S) based on their mean disease
ratings. Cluster 1 (purple), Cluster 2 (light yellow), Cluster 3 (sky blue), Cluster 4 (steel
blue). A white space and black tick marks separate subgroups of individuals.

#$!

!

Figure 3.5. Population structure grouped by Phytophthora fruit rot resistance to isolate
12889 at A) 3 days post inoculation and B) 5 days post inoculation. Individuals were
grouped into a resistant and moderately resistant category (R/MR), a moderately
susceptible category (MS), and a susceptible category (S) based on their mean disease
ratings. Cluster 1 (purple), Cluster 2 (light yellow), Cluster 3 (sky blue), Cluster 4 (steel
blue). A white space and black tick marks separate subgroups of individuals

A total of 102 alleles were detected among the 23 SSRs, evaluated in the
collection, ranging from 2 to 7 alleles per locus with an average allele diversity of 4.9
alleles per locus. The mean genetic diversity index of the collection was 0.44. The mean
polymorphism information content (PIC) value was 0.40 for the collection and individual
markers ranged from 0.12 to 0.71 for the population (Table 3.2). When individual
genotypes were evaluated, the highest PIC value was 0.54 in PI 640809 and the lowest
PIC value was 0.03 in lines JN566, PI 148628, and PI 640803. Genetic diversity was
similarly distributed within continents (0.38-0.47), and pairwise Fsts indicated little to
moderate differentiation between continents (0.00 – 0.07) (Table 3.6, Table 3.7).
#%!

!

Table 3.6. Genetic diversity of pepper genotypes among countries and continents
b
Diversity Estimates
a

Category
Africa
Asia
Europe
N. America
S. America
a

GD
PIC
0.40
0.36
0.44
0.39
0.38
0.34
0.47
0.43
0.41
0.37
b
Diversity Estimates

Category
GD
PIC
Afghanistan
0.28
0.23
Brazil
0.35
0.27
China
0.35
0.29
Colombia
0.27
0.22
Former Soviet
0.35
0.29
India
0.34
0.29
Iran
0.43
0.38
Mexico
0.45
0.41
Pakistan
0.36
0.30
Serbia
0.26
0.22
Spain
0.27
0.22
Turkey
0.36
0.31
a
Categories represented by less than four lines were excluded from analyses and are not
shown
b
Average values for SSRs are presented; Mean values are presented for the genetic
diversity (GD) and the polymorphism information content (PIC)

$&!

!

Table 3.7. Genetic differentiation (FST pairwise differentiation) of pepper genotypes among countries and continents
b

a

Category
Brazil
China
Colombia
Former
Soviet
India
Iran
Mexico
Pakistan
Serbia
Spain
Turkey
USA

Brazil

China

0.19*
0.20

0.19

0.20
0.25*
0.24*
0.37*
0.19*
0.23
0.11
0.32*
0.35*
a

Afghanistan
0.18
0.26*
0.24

0.14
0.17
0.19*
0.23*
0.17*
0.20
0.20
0.21*
0.24*

0.17*
0.17*
012*
0.18*
0.16*
0.17
0.19
0.18*
0.16*
b
Fst

Colombia

Fst
Former
Soviet India

0.09
0.09
0.15
0.28*
0.13
0.16
0.09*
0.16
0.21*

0.06
0.06
0.14*
0.13*
0.09
0.11
0.06
0.13*

0.10*
0.13*
0.14
0.21
0.13
0.07
0.08

Iran

0.06*
0.11*
0.11
0.18*
0.00
0.08*

Mexico Pakistan Serbia Spain

0.22*
0.27*
0.33*
0.07*
0.05*

0.16
0.19
0.11
0.21*

0.19
0.22
0.25*

0.25
0.29*

Category
Africa
Asia Europe
N. America
Asia
0.06
Europe
0.02
0.02
N. America
0.07*
0.00*
0.03
S. America
0.01
0.05*
0.01
0.06*
a
Categories with less than four lines were excluded from analyses and are not shown
b

Average values for SSRs are presented; * indicates value was outside the 2.5% and 97.5% confidence intervals at 1000
bootstraps

"#!

!

Individuals from N. America, Asia and S. America had the highest genetic diversity and
genetic differentiation was highest in individuals from N. America. Genetic diversity
within countries varied greatly (0.26 – 0.45) and pairwise Fst values detected little to very
great genetic differentiation among countries (Table 3.6, Table 3.7). Genetic diversity
was highest in individuals from Iran, Mexico and the USA, the greatest differentiation
was seen with individuals from Mexico and the USA. Pairwise Fsts for disease resistance
to 12889 and OP97 showed little to great (0 to 0.22) genetic differentiation between
categories (Table 3.8).
Table 3.8. Genetic differentiation (FST pairwise differentiation) among pepper disease
resistance categories 3 (shaded values) and 5 days post inoculation (dpi) when inoculated
with OP97 and 12889
Isolate OP97
Category
MR
MS
R
S

a

Pairwise Fst
MR
0.01
0.01
0.05

MS
0.18
0.01
0.04

b

R
-

S
0.50*
0.00*
-

0.04

Isolate 12889
a

Category
MR
MS
R
S
MR
0.01*
0.01
0.02*
MS
0.12*
0.03
0.01*
R
0.03
S
0.49
0.00*
a
3 dpi are shaded above the diagonal and 5dpi values are below the diagonal for OP97
and 12889; MS = moderately susceptible, R = resistant MR = moderately resistant,
S=susceptible
b
Average values for SSRs are presented; * indicates value was outside the 95%
confidence interval at 1000 bootstraps

"#!

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When inoculated with isolate OP97, no significant genetic differentiation was evident
between any of the categories at 3dpi. At 5dpi, individuals in the resistant/moderately
resistant (R/MR) category and the susceptible (S) category were significantly
differentiated, as were individuals in the moderately susceptible (MS) and R/MR
categories. When inoculated with 12889, differentiation between categories was
significant at 3dpi for MS and MR categories, and MS and S categories. Significant
differentiation was detected at 5dpi between the MR and S, MS and S and the MR and
MS categories at 3dpi when inoculated with OP97.

DISCUSSION
Pepper is an important crop grown and cultivated around the world and P. capsici
is a devastating pathogen that causes economic losses annually. In this study, we
evaluated Phytophthora fruit rot resistance, population structure and genetic diversity of a
diverse collection of peppers from around the globe. Significant differences among lines
and countries were detected for disease resistance. Significant population structure was
detected when grouped by predefined categories of disease resistance, country and
continent of origin.
Most accessions evaluated in this study were highly susceptible to both isolates of
P. capsici at 5dpi, while many displayed partial resistance at 3dpi. At 5dpi, disease
incidence varied greatly among lines, and lines with partial resistance, in general, had
lower incidence than the most susceptible lines. The resistant control, CM334, had a high
incidence of disease, and the mean lesion area was moderately susceptible, 14 and 17
2

cm , depending on the isolate. The susceptible control, JN571, also had a high incidence
"$!

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2

of disease, and lesion area was high (~50 cm ). Several individuals were identified with
higher resistance than CM334 and are potential sources of resistance for breeding
programs. Inoculations were made under ideal disease conditions, and those lines with
lower incidence may prove to be resistant under field conditions. No lines evaluated
were completely resistant to Phytophthora fruit rot at 5dpi, this included lines with known
resistance to Phytophthora root rot (18). Markers known to segregate for P. capsici root
rot were also not informative when searching for population structure related to fruit
disease resistance (76). These results are consistent with previous reports that found no
associations between fruit and root rot (31,111).
Certain geographic regions were significantly less susceptible than others
(countries and continents). In particular, fruit from Serbia were the most susceptible and
fruit from Pakistan were the least susceptible at 3 and 5dpi. Country and continent were
not predictive of resistance however, with all countries and continents contributing very
2

susceptible, 20 cm or greater lesion area at 5dpi, individuals. Mexico and S. America,
the center of origin for C. annuum and some Phytophthora spp. (14,39) were among the
more resistant countries/continents. These results were not unexpected, since most pepper
lines with resistance to P. capsici-induced diseases are landraces from Mexico and
Mexico is known to be the center of origin for C. annuum.
Genetic diversity within this collection of C. annuum was moderate (0.44), and
most individuals were grouped into 1 of 4 genetic clusters based on the Bayesian
analysis. Clusters did not completely correspond with predefined categories of continent,
country, or disease susceptibility though some clusters were better represented in
categories than others. Few individuals (15 of 157) were admixed, consistent with
"%!

!

previous results (1,51,65). C. frutescens and C. chinense individuals were grouped within
cluster 3 as were several C. annuum lines. These C. annuum lines in cluster 3 were
predominantly from S. America and are likely the result of introgression or
misclassification. Many of these individuals had growth habits or flower color
reminiscent of C. frutescens, C. baccatum or C. chinense (data not shown). These data
suggest limited introgression from related species into individuals of C. annuum within
available germplasm, and minimal cross pollination among genetic clusters.
When based on geographic regions, populations in Asia and North America were
represented by individuals from each C. annuum cluster. In Asia, the small-fruited lines
and landraces commonly grown may be more akin to ancestral populations and may be
the result of selection for different local markets. In N. America the high diversity and
broad population profile is likely due to the inclusion of different market classes (sweet
fruited bells and pungent chiles) and breeding lines in the population (51,87). Individual
countries in Asia did not display the diversity seen across the continent. The USA (the
only country represented by four individuals in N. America) was represented by multiple
market classes and maintained this diverse population profile seen for N. America.
Phytophthora fruit rot resistance, population structure, and genetic diversity
demonstrated significant differences between countries, and continents in this diverse
collection of peppers. Disease susceptibility was significantly associated with population
structure, continents and countries of origin at both 3 and 5dpi. These results provide the
groundwork for Phytophthora fruit rot resistance, genetic diversity and population
structure of Capsicum annuum on a global scale. Further work is needed to identify
pepper lines with full resistance to Phytophthora fruit rot.

"&!

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BIBLIOGRAPHY

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BIBLIOGRAPHY
)*!+,-./01234/456478!+*8!39114//8!:*!;*8!<99=48!3*!;*8!056!>.?8!@*2A*!#BBC*!D454E.F!
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K!"#$%&'()"**''();*M!,459EHX4=!14G40/46!VH!+Y;:!050/H=4=*!+N1*!P*!
Q.9E4FJ59/*!"!
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6.G41=.EH!.5!!"#$%&'()+"&&",'(!.=!=.,5.N.F05E/H!.5N/-45F46!VH!.E=!
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