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Michigan State
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This is to certify that the

thesis entitled

Genetic Diversity of Prunus Serotinaand the
Evaluation of Other Wild Species for Breeding
Sour Cherry Resistance to Cherry Leaf Spot

 

presented by

Suzanne Lynn Downey

has been accepted towards fulfillment
of the requirements for

 

Masters of Scienchegree in Plant Breeding and Genetics
— Horticulture

(ZW (Wm
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Date February 14 1999

0-7639 MS U is an Affirmative Action/Equal Opportunity Institution

 

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GENETIC DIVERSITY OF PR UNUS SEROTINA AND
THE EVALUATION OF OTHER WILD SPECIES FOR BREEDING
SOUR CHERRY RESISTANCE TO CHERRY LEAF SPOT
By

Suzanne Lynn Downey

A THESIS
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE
Plant Breeding and Genetics
Department of Horticulture

1999

Professor Amy F. Iezzoni

ABSTRACT
GENETIC DIVERSITY OF PRUNUS SEROTINA AND THE EVALUATION OF
OTHER WILD SPECIES FOR BREEDING SOUR CHERRY RESISTANCE To
CHERRY LEAF SPOT
By

Suzanne Lynn Downey

Central to the cherry breeding program at MSU is the acquisition and screening of
germplasm for the development of improved sour cherry cultivars. Breeding for disease
resistance to cherry leaf spot caused by Blumeriellajaapii depends upon the identification
of resistance. The following germplasm was screened to identify B. jaapii resistance: “GI
148-2, GI 148-1, MxM2, MxM60 and seedlings of wild black cherry (Prunus serotina).
MxM2, MxM60 and GI 148-2 were highly susceptible to B. jaapii. GI 148-1 was
resistant, exhibiting no sporulation, chlorosis, or defoliation. This selection is currently
being used as a donor of leaf spot resistance in a backcross breeding program. P.
serotina, immune to B. jaapii in our study, is of particular interest because of the
occurrence of large fruited types. To gain a baseline understanding of the genetic diversity
within P. serotina from Michigan, Mexico and Ecuador, the following informative
marker systems were utilized. Three chloroplast polymorphisms were identified with the
most common fragment present in all three geographic groups. Putative allozymes were
identified in each geographic group. Sequence tagged site markers revealed the most
genetic diversity. Mexican P. serotina germplasm had the largest number of putative
alleles. The data suggests that the Michigan and Ecuadorian gerrnplasms are divergent,

most likely due to ecological and geographic adaptation and domestication events.

DEDICATION

This work is dedicated to my family, whose constant love and support have made
this all possible. Thank you for believing in me and constantly reminding me of those

things that are truly important in life. . .all of you.

ACKNOWLEDGEMENTS

Without the constant support of my major professor, Dr. Amy Iezzoni, this
accomplishment would not be possible. I sincerely thank her for understanding, support
and never ending encouragement which helped me to develop my own research interests.
Dr. Jim Hancock was not only instrumental in my education and research, but he is also
one of the primary reasons why I pursued my MS at Michigan State University through
the Plant Breeding and Genetics Program. A big thanks goes out to him, as well as the
other members of my committee, Dr. Alan Jones and Dr. Bryan Epperson. Dr. Salvador
Perez from the Universidad de Queretaro, Mexico, was essential to my research. He was
key for the germplasm collection trip into central Mexico and remains the primary P.
serotina collaborator for our lab. Dr. Albert Abbott and Graham King were our sweet
cherry and peach derived primer pair sources and also deserve recognition. The
successful completion of my MS would not have been possible without the love and
support of the friends I have made during my stay at MSU, especially those people who
are part of our extended laboratory and my extended ‘family.’ Thank you Renate Karle,
Pete Callow, Dechun Wang, Erin Crowe, Christopher Owens, Rebecca Henry, Jaimie
Houghton, Audrey Sebolt, Kirsten and Soren Ottosen, Roger Herr, Charlie Herman, Beth
Faussey, the ladies of Alpha Omicron Pi, and Leslie Finical. You have made all the

difference.

iv

TABLE OF CONTENTS

LIST OF TABLES ................................................................................. vi
LIST OF FIGURES ................................................................................ Vii
CHAPTER ONE

EVALUATION OF WILD SPECIES FOR BREEDING SOUR CHERRY (PR UN US
CERAS US) RESISTANCE TO CHERRY LEAF SPOT CAUSED BY BL UMERIELLA

JAAPII .................................................................................................. 1
Abstract ....................................................................................... 2
Introduction and Literature Review ....................................................... 3
Materials and Methods ...................................................................... 8
Results ....................................................................................... 11
Discussion ................................................................................... 17
Conclusions ................................................................................ E. 18
Literature Cited ............................................................................. 19

CHAPTER TWO

GENETIC DIVERSITY WITHIN AND AMONG BLACK CHERRY (PR UN US

SEROT INA) FROM MICHIGAN, MEXICO AND ECUADOR .............................. 23
Abstract ...................................................................................... 24
Introduction and Literature Review ...................................................... 25
Materials and Methods ..................................................................... 31
Results ....................................................................................... 35
Discussion ................................................................................... 56
Conclusions ................................................................................. 59
Literature Cited ............................................................................. 60

APPENDIX A: COLLECTION INFORMATION FOR MEXICAN P. SEROT INA

SELECTIONS .............................................................................. 65
APPENDIX B: ECUADORIAN GERMPLASM .............................................. 72
APPENDIX C: PRIMER PAIR SOURCES AND SEQUENCES ........................... 75
APPENDIX D: RAW STS DATA ............................................................... 79

LIST OF TABLES

Table 1. Comparison of the mean number of necrotic lesions per cm2 for sour cherry
(P. cerasus), GI 148-1, and GI 148-2 fourteen days after artificial inoculation
with B. jaapii conidia ..................................................................... 13

Table 2. A summary of disease symptoms exhibited by sour cherry, GI 148-1 and
GI 148-2, MxM hybrids, and P. serotina selections following artificial
inoculation with B. jaapii conidia ........................................................ 14

Table 3. Number of open-pollinated P. serotina seedlings per family assayed for
chNA fragments, 6-PGD, PGI, and sequence tagged sites (STSs) ................ 41

Table 4. Optimized PCR conditions for each primer pair used to amplify a chloroplast
non-coding region and nuclear sequence tagged sites (STSs) in P. serotina
from Michigan, Mexico, and Ecuador .................................................. .42

Table 5. Fragments amplified from successive PCR reactions on a chloroplast non-
coding region [AB] from P. serotina selections from Michigan, Mexico and
Ecuador ...................................................................................... 43

Table 6. Summary of informative and non-informative primer pairs derived from
sweet cherry and peach on P. serotina selections fi'om Michigan, Mexico
and Ecuador ................................................................................. 44

Table 7. Collection information for Mexican P. serotina selections ........................ 67

Table 8. Ecuadorian P. serotina germplasm identification and germination
information .................................................................................. 74

Table 9. Primer pair sources and sequences .................................................... 77

Table 10. Data matrix for primer pair GA 34 containing 14 putative alleles
numbered according to their corresponding fragment lengths for each of the
66 P. serotina selections assayed ......................................................... 81

Table l 1. Data matrix for primer pair P812A02 containing 12 putative alleles
numbered according to their corresponding fragment lengths for each of the
66 P. serotina selections assayed ......................................................... 85

Table 12. Data matrix for primer pair B6B] containing 19 putative alleles

numbered according to their corresponding fragment lengths for each of the
66 P. serotina selections assayed ......................................................... 89

vi

Table 13. Data matrix for primer pair BlOH3 containing 9 putative alleles
numbered according to their corresponding fragment lengths for each of the
66 P. serotina selections assayed ......................................................... 95

vii

LIST OF FIGURES

Figure 1. Cherry leaf spot disease on the (A) adaxial and (B) abaxial leaf surface
of GI 148-1, Gl 148-2, and sour cherry selections fourteen days after initial
inoculation with B. jaapii conidia ........................................................ 15

Figure 2. (A) Leaves of sour cherry and GI 148-1 selections infected with B. jaapii
conidia. (B) German hybrid GI 148-1 exhibiting few necrotic lesions, no
chlorosis or leaf abscission 21 days after initial inoculation with B. jaapii
conidia ....................................................................................... 16

Figure 3. Amplified non-coding regions of P. serotina chloroplast genome resolved
on 6% polyacrylamide gels and stained with Silver SequenceTM Stain. Gels
are oriented with loading origin at the top of each photograph ...................... 45

Figure 4. Pgi-l and Pgi-2 loci patterns in P. serotina samples from Michigan,
Mexico, and Ecuador. Gels are oriented with the sample loading origin at
the bottom of each photograph. Selected loci assigned alleles and out groups
GI 148-1 and GI 1482 are labeled with arrows ........................................ 46

Figure 5. Zymograrn representing patterns exhibited by P. serotina selections from
Michigan, Mexico and Ecuador and out group controls assayed for
Phosphoglucose isomerase (PGI). Alleles I represent putative alleles for P.
serotina. Alleles II represent putative alleles for out group controls.

Pattern 1: Michigan (MI), Mexican (U2, ARG, P3, RG, U3, J 1, Pl, TI, PH,

and J3), and Ecuadorian (Ecu A-H) P. serotz‘na and EB (Erdi Botermo).

Pattern 2: Michigan (MI), Mexican (P2, J 1, Pl , TI and U3), and Ecuadorian

P. serotina (Ecu A-H). Pattern 3: Michigan (MI), Mexican (U3 and RG) and
Ecuadorian P. serotina (Ecu A-H). Pattern 4: Emperor Francis (EF),

Csengodi, Montrnorency, and Rheinische Schattenmorelle (RS). Pattern 5:

GI 148-1 and GI 148-2 ..................................................................... 47

Figure 6. 6-Pgd-1 and 6-Pgd-2 loci patterns in P. serotina samples from Michigan
Mexico, and Ecuador. Gels are oriented with the sample loading origin at
the bottom of each photograph. Selected loci assigned alleles are labeled
with arrows .................................................................................. 48

Figure 7. Zymograrn representing banding patterns exhibited by P. serotina selections
from Mexico and Ecuador and out group controls assayed for
6-phosphogluconate dehydrogenase (6-PGD). Alleles I represent putative
alleles for P. serotina. Alleles II represent putative alleles for out group
controls. Banding pattern 1: Mexican (TI, P2, P1, P3, U2, U3, ARG, J3,

viii

PH, RG, and J1) and Ecuadorian (Ecu A-H) P. serotina accessions. Pattern 2:
Mexican (P1, ARG, J3 and J1) and Ecuadorian (Ecu A-H) accessions.

Pattern 3: Mexican (P3 and P1) and Ecuadorian (Ecu A-H) accessions.

Pattern 4: Emperor Francis (EF). Pattern 5: Csengodi and GI 148-2.

Pattern 6: GI 148-1, Montmorency, Rheinische Schattenmorelle (RS) and

Erdi Botenno (EB) ......................................................................... 49

Figure 8. Photographs of 6% polyacrylamide gels resolving informative STS markers
from sour and sweet cherry. Putative alleles for Michigan, Mexico and
Ecuador P. serotina accessions resulting from amplification with primer pair
GA 34 (A) and primer pair P812A02 (B) ............................................... 50

Figure 9. Putative alleles identified by STS GA34 for Michigan, Mexico and Ecuador
P. serotina selections. Putative allele numbers correspond to amplified band
sizes (bp) .................................................................................... 51

Figure 10. Putative alleles identified by STS P812A02 for Michigan, Mexico and
Ecuador P. serotina selections. Putative allele numbers correspond to .
amplified band sizes (bp) .................................................................. 52

Figure 11. Photographs of informative STS marker systems from peach resolved
on 6% polyacrylamide gels. Putative alleles for Michigan, Mexico and
Ecuador P. serotina accessions resulting from amplification with primer pair
B6B] (A) and BlOH3 (B&C) ............................................................ 53

Figure 12. Putative alleles identified by STS B6B1 for Michigan, Mexico and
Ecuador P. serotina selections. Putative allele numbers correspond to
amplified band sizes (bp) .................................................................. 54

Figure 13. Putative alleles identified by STS BlOH3 for Michigan, Mexico and
Ecuador P. serotina selections. Putative allele numbers correspond to
amplified band sizes (bp) .................................................................. 55

ix

CHAPTER ONE

EVALUATION OF WILD SPECIES FOR BREEDING SOUR CHERRIES
(PR UN US CERAS US) RESISTANT TO CHERRY LEAF SPOT CAUSED BY

BL UMERIELLA JAAPII

ABSTRACT

Cherry leaf spot, caused by Blumeriellajaapii (Rhem) Arx., is a major fungal
disease of sour cherry (Prunus cerasus L.) affecting production orchards worldwide. All
principal sour cherry cultivars grown are susceptible to B. jaapii and numerous fungicide
sprays are required to prevent this disease. One of the objectives of the sour cherry
breeding program at MSU is to breed cultivars resistant to cherry leaf spot. To
accomplish this goal, identification of suitable donor species of increased disease
resistance is required. Previous work suggested that the best source(s) of resistance may
be wild Prunus species and interspecific hybrids. Therefore, the following germplasm
was obtained and screened for increased disease resistance: GI 148-1 and GI 148-2 (P.
cerasus x P. canescens), MxM2 and MxM60 (P. mahaleb x P. avium), and open-
pollinated seedlings of wild black cherry (P. serotina). Sour cherry seedlings were used
as susceptible controls. Young leaves of each selection were inoculated with a fresh
spore suspension of B. jaapii collected from naturally infected sour cherry leaves and
disease progression was evaluated 14 days after inoculation. P. serotina was immune to
leaf spot with no apparent infection. Sour cherry, MxM2, MxM60, and GI 148-2 were
susceptible to B. jaapii, all exhibiting necrotic lesions, fungal sporulation, leaf chlorosis
and defoliation. In contrast, GI 148-1 was resistant to B. jaapii infection exhibiting
necrotic lesions that eventually excised, no fungal sporulation, and no leaf chlorosis or
defoliation. GI 148-1 also had significantly fewer necrotic lesions per cm2 than
susceptible sour cherry controls. Currently, GI 148-1 is being used as a donor of leaf spot

resistance in the MSU sour cherry breeding program.

INTRODUCTION AND LITERATURE REVIEW

The Disease

Blumeriellajaapii (Rhem) Arx. (syn. C accomyces hiemalis Higgins) causes a
major disease of sour cherry (P. cerasus) commonly referred to as cherry leaf spot or
‘shot hole’ disease. Unfortunately, the principal sour cherry variety grown within the
United States, ‘Montmorency,’ is highly susceptible to this disease (Andersen, 1981). As
a result, a major focus of the sour cherry breeding program at MSU is the development of
cultivars resistant to cherry leaf spot disease.

The fungus causing cherry leaf spot was first reported in Europe during 1884 on i
P. padus (Karsten, 1884). Not until the work of Higgins (1914) was the life cycle of the
fungus understood. Higgins (1914), after associating the sexual and asexual stages
together and describing the life cycle, classified the fungus causing cherry leaf spot into
three distinct species according to host range: C. hiemalis found on P. avium, P. cerasus,
and P. pennsylvanica; C. lutescens found on P. serotina, P. virginiana, and P. mahaleb;
and C. prunophorae found on plums (P. americana, P. domestica, and P. insititia).
After several revisions of the taxonomy, the fungus was eventually named B. jaapii
(Rhem) Arx., pooling all European and American species together as one (von Arx,
1961).

When not properly controlled, cherry leaf spot disease can cause petiole and leaf
chlorosis and premature defoliation. In these instances, fruit that fail to ripen are poorly

colored and have low soluble solids (Jones, 1976). Premature defoliation also contributes

to an overall reduction of tree vigor due to reduced flower bud and wood survival during
winter months (Howell and Stackhouse, 1973).

The disease cycle begins each year in the spring when the overwintered fungus
produces mature apothecia. Ascospores are released upon wetting and are disseminated
from lingering leaf debris on the orchard floor to young cherry leaves primarily through
wind and splashing rain (Higgins, 1914). When proper weather conditions exist,
ascospore dispersal can continue from the end of cherry bloom until June (Keitt et al.,
1937). Primary inoculation of cherry leaves begins when ascospores enter through open
stomates on the abaxial leaf surface (Keitt et al., 193 7; Jones et al., 1993; Garcia et
al.,1993). Young unfolded leaves are the most susceptible to inoculation (Eisensmith et
al. 1982). According to Eisensmith and Jones (1981), disrupted periods of wetness may
be more favorable for severe primary infection on young cherry leaves than continuous
wet periods. Once infection has occurred, fungal hyphae grow throughout the host leaf
tissue causing the leaf to form necrotic lesions.

Disease severity progresses as the fungus advances throughout susceptible host
leaf tissue. Severe disease symptoms include necrotic lesions which do not excise,
considerable leaf and petiole chlorosis, and leaf abscission (Higgins, 1914; Jones 1976).
A sign of disease progression is the eventual production of white acervuli on the abaxial
leaf surface. Mature acervuli sporulate during periods of high humidity, releasing conidia
which subsequently disseminate to new host tissue through water droplets and air currents
(Higgins, 1914). Without control measures, secondary infection can occur readily
throughout the cherry growing season, causing widespread orchard devastation. This

secondary spread of the disease is dependent upon the amount of inoculum available and

the occurrence of favorable temperature and moisture conditions (Eisensmith and Jones,
1981). B. jaapii overwinters during its sexual phase in protective stroma-like structures
which develop on infected cherry leaves on the orchard floor (Higgins, 1914).

Removal of infected leaf debris is critical to the reduction of primary inoculum
and delaying disease infection in subsequent years (Keitt et al., 193 7). F ungicide
regiments of four to six sprays per year are the most common defense against the spread
of B. jaapii in orchards (Jones and Ehret, 1980). Highly effective infection models for
timing of fungicide applications have been developed to reduce unnecessary and
ineffective sprays (Eisensmith, 1981). However, alternative control measures are needed
since the fungicides used to control cherry leaf spot are costly and there is concern about.
the occurrence of fungicide resistance in B. jaapii (Jones and Ehret, 1980; Jones et al.,
1993). Identification of natural disease resistance in closely related Prunus species is
therefore a major part of the strategy to fight this disease.

Natural Disease Resistance in Prunus

The development of disease resistant varieties of sour cherry is a major priority of
the Michigan State University sour cherry breeding program. The sour cherry variety
‘Montmorency’ is highly susceptible to cherry leaf spot disease. P. cerasus is a
tetraploid species (2n = 4x = 32) believed to be the result of interspecific hybridization
between sweet cherry (P. avium L.) and European ground cherry (P. fi'uticosa Pall.)
(Olden and Nybom, 1968). All three species readily intennate and sour cherry exhibits
traits from both parental species. This intennixing has created natural diversity from

which to select individuals for superior fruit quality and adaptive traits (lezzoni et al.,

1 990).

Initially, resistance was sought in closely related Prunus species. Sweet cherry,
sour cherry, European ground cherry, and duke cherry (P. gondouinii Rehd.) and ‘North
Star’ varieties which appeared to have field resistance to this disease were screened under
laboratory conditions (Sjulin et al., 1989). If identified, resistance in these closely related
species could readily be transferred into sour cherry. However, all screened selections
were susceptible to B. jaapii. Sporulation and subsequent spread of the disease was
reduced in some selections. As a result, breeding efforts are currently focused on
identification of resistance in interspecific hybrids and wild Prunus species.

Existing interspecific hybridizations between sour cherry and selected Prunus
species may be a good source of cherry leaf spot resistance. Commercialized dwarfing '
rootstocks for sweet cherry, GI 148-2 (Giesla 5) and GI 148-1 (Giesla 6), are German
hybrids resulting from the cross between P. cerasus cv. Schattenmorelle and P. canescens
(Schmidt and Gruppe, 1988). Since P. canescens is a diploid species, both hybrids are
triploid. Schattenmorelle, the sour cherry parent, is highly susceptible to cherry leaf spot,
whereas the P. canescens parent used to produce these hybrids has been observed to be
immune to the disease (E. Gigadlo, pers. communication). It is possible that these full
sibling hybrids may have inherited some disease resistance from their P. canescens
parent.

Other commercially available cherry rootstocks (MxM2 and MxM60) are from
presumed natural hybridizations originating in Oregon between P. mahaleb and P. avium
(P. mazzard) (Cummins, 1979). These selections have been previously documented as

exhibiting a variety of reactions to B. jaapii ranging from resistant to severely susceptible

(Westwood, 1976). Screening of this germplasm is necessary to evaluate these hybrids as
donor species of increased disease resistance.

Another potential source of disease resistance to cheny leaf spot may be wild
black cherry (P. serotina Ehrh). Commercially grown P. serotina within the United
States have been documented to be susceptible to one specific race of B. jaapii, C.
Iutescens Higgins (Higgins, 1914; Heald, 1933; Davis, et al. 1942; Stanosz, 1992). P.
serotina of the US, used for its high valued lumber, has been overlooked by cherry
breeders as a source of resistance due to its production of extremely small fruit (aprox.
0.5 cm in diameter). However, large fruited (avg. fruit 2 cm in diameter) varieties of P.
serotina called “Capulin” exist in Central Mexico and Ecuador (Popcnoe and Pachano, I
1922; Popenoe, 1924). Little information regarding the resistance of large fruited wild
populations of P. serotina spp. capuli to B. jaapii is available. P. serotina is a tetraploid
species (2n = 4x = 32, Stairs and Hauck, 1968) making it a good candidate for possible
incorporation of disease resistance into sour cherry if superior resistance to B. jaapii is
identified. Therefore, screening of Capulin selections with fruit characteristics more
similar to the desired fruit size in commercial sour cherry cultivars is necessary
investigate their potential as donors of leaf spot resistance in the MSU breeding program.
Objective

The objective of this study was to evaluate the following selections as sources of
cherry leaf spot resistance: GI 148-1, GI 148-2, MxM2, MxM60, and wild black cherry

selections from Mexico and Ecuador.

MATERIALS AND METHODS

Plant Material

Rooted cuttings of GI 148-1 were obtained form Hilltop Nurseries (Hartford, MI).
Seeds of wild black cherry were collected in Central Mexico and Ecuadorian accessions
were obtained from the Ecuadorian Gerrnplasm Institute (Appendix A, B). The seeds
were germinated and the resulting plants were grown in a MSU growth chambers and
greenhouse. P. serotina seedlings, along with hybrid cuttings and sour cherry seedlings
resulting from seed harvested in 1995, were transplanted and grown in a completely
randomized design within a plastic greenhouse at the Michigan State University
Clarksville Horticultural Research Station (CHES) in Clarksville, Michigan. In August
of 1996, five GI 148-1 cuttings, five P. serotina seedlings from Mexico and Ecuador, and
five sour cherry seedlings were randomly selected for inoculation. The experiment was
repeated in 1997 with the addition of interspecific hybrids GI 148-2 , MxM2 and
MxM60. Rooted cuttings of these selections were also obtained from Hilltop Nurseries
(Hartford, MI).
Inoculation

Fresh inoculum was obtained for the 1996 and 1997 inoculations from naturally
infected leaves of sour cherry collected from an unsprayed, infected sour cherry orchard
(CHES). Infected leaf samples were placed in a moist plastic bag at 4°C until the time of
inoculum preparation. To maximize spore germination and infection, overhead sprinklers
were run in the plastic greenhouse for at least one hour prior to inoculation to increase

humidity and ensure opening of the leaf stomates. Conidial suspensions of B. jaapii were

prepared by washing the infected leaves with water and carefully brushing off visible
acervuli with a clean paint brush. After a cloudy inoculum slurry was obtained, the
suspension was cleaned of debris by filtration through two layers of cheesecloth. The
conidial inoculum was then transferred into a small clean spray bottle. A quantitative
inoculator like that developed by Schein (1964) was not used since such equipment was
not available and precise quantitation of inoculum was not deemed necessary for an initial
disease assessment. In addition, identification of inoculum races was not performed.
Seedlings were inoculated by ‘uniformly’ spraying the abaxial leaf surface of the second
unfolded leaf of a vigorously growing shoot three times on a fine mist setting. The 1996
and 1997 inoculations were performed during the overcast, late afternoons of August 29 E
and August 20, respectively. The inoculum slurry was also sprayed onto potato dextrose
agar (PDA) plates and subsequently examined under 200x magnification after 24 hours of
incubation at room temperature to confirm germination of conidia.
Disease Evaluation

Fourteen days after initial inoculation, necrotic lesions were counted on five
leaves of each inoculated plant. Observations of disease symptoms and progression were
also periodically recorded for three weeks following initial inoculation. On day fourteen,
leaf area for each evaluated leaf was determined. To be sure not to interfere with disease
progression, inoculated leaves were not harvested from the selections. Instead, leaf
outlines were traced on paper and used for calculating total leaf area with a leaf area
meter (Li Cor, Lincoln, Nebraska). The number of lesions per cm2 was calculated for
each of the infected leaves as the number of lesions on day fourteen divided by the leaf

area. The average of five replications for each selection was calculated. Statistical

comparisons among varieties were performed using Proc GLM in SAS (SAS Institute,

1989).

10

RESULTS

Conidial Germination

Conidial genuination was defined as the stage at which the germ tube was twice
as long as the conidia. In both 1996 and 1997, the spores used in inoculation experiments
exhibited 80% germination.
Disease Susceptibility Evaluation

In 1996 and 1997, GI 148-1 exhibited 1.6 and 1.2 necrotic lesions per cm2
respectively (Table 1). The numbers of necrotic lesions for GI 148-1 were significantly
less than those exhibited by the susceptible sour cherry controls which averaged 19.4 and
5.4 necrotic lesions per cm2 in 1996 and 1997, respectively (P = 0.05). GI 148-2
averaged 8.2 necrotic lesions per cm2 in 1997. Although this result was not significantly
different from the susceptible sour cherry controls, GI 148-2 had significantly more
necrotic lesions per cm2 than its full sibling, GI 148-1 (P = 0.05).

Sour cherry and GI 148-2 exhibited similar symptoms of susceptibility to cherry
leaf spot (Table 2). Leaves from these selections became chlorotic while those of GI 148-
1 failed to Show symptoms of leaf or petiole chlorosis (Figure 1). In addition to
producing relatively high lesion counts per cmz, sour cherry controls and GI 148-2 also
exhibited visible white acervuli containing conidia on the abaxial leaf surface and leaf
abscission within 21 days of initial inoculation (Table 2, Fig. 2). In contrast, GI 148-1
exhibited no apparent sporulation, leaf chlorosis, or defoliation (Table 2, Fig. 2, Fig. 3,
Fig. 4). Necrotic lesions found on G1 148-1 were small necrotic circles (~2mm diameter)

which eventually excised (Fig. 4), producing characteristic ‘shot hole’ symptoms. When

inoculated leaves of GI 148-1 exhibiting necrotic lesions were-removed from the trees
and placed in optimal sporulation conditions (high humidity to force conidial release), no
sporulation was observed.

MxM2 and MxM6O were susceptible to cherry leaf spot disease when inoculated
with B. jaapii from naturally infected sour cherry leaves (Table 2). Necrotic lesion
calculations were not taken for MxM2 and MxM60 selections because of the severity of
disease symptoms exhibited by each hybrid.

P. serotina seedlings failed to exhibit any disease symptoms when inoculated with
the spore suspension of B. jaapii isolated from infected P. cerasus (Table 2). Black cherry
seedlings included in this study exhibited no signs of infection or symptoms of cherry leaf

spot disease.

12

Table 1. Comparison of the mean number of necrotic lesions per cm2

for sour cherry (P. cerasus), GI 148-1, and GI 148-2 fourteen
days after artificial inoculation with B. jaapii conidia.

 

 

Cherry Average number of
Year selection lesions per cm2
1996 Sour cherry 19.4y a2

GI 148-1 1.6 b
1997 Sour cherry 5.4 a

GI 148-1 1.2 b

GI 148-2 8.2 a

 

y Each value represents the mean of 5 replications.
2 Within year, means followed by different letters are
significantly different at P = 0.05

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53» cote—soofi .3055 wage—Em 82828 32.22%. .k 98 £2.53 2x2
.3: .o as E: 6 .923 58 B 3525 Baasa 083% co bases... < .m 2.3

14

\ .

Em I48-l Sour(‘hcrr} ,(.‘l 148-2

{CI 143-1 Mum hem (.‘l I48-2

 

Figure 1. Cherry leaf spot disease on the (A) adaxial and (B) abaxial leaf
surface of GI 148-1, GI 148-2, and sour cherry selections fourteen
days after initial inoculation with B. jaapii conidia.

Sour (‘herry " (:1 148-1

 

B

Figure 2. (A) Leaves of sour cherry and GI 148-1 selections infected with
B. jaapii conidia. (B) German hybrid GI 148-1 exhibiting few
necrotic lesions, no chlorosis or leaf abscission 21 days after initial
inoculation with B. jaapii conidia.

DISCUSSION

GI 148-1 was identified as a source of resistance to cherry leaf spot since
inoculation with B. jaapii resulted in small necrotic lesions that failed to sporulate. The
characteristic shot hole symptoms exhibited by GI 148-1 prevented sporulation and
further spread of B. jaapii to new host tissue. According to Parlevliet (1979), GI 148-1
exhibits a disease reaction defined as infection type zero (ITO), where necrotic symptoms
fail to sporulate.

The sour cherry parent of GI 148-1, P. cerasus Schattenmorelle, is highly
susceptible to cherry leaf spot whereas P. canescens has been observed to be immune to
the disease (E. Gigadlo, pers. communication). Therefore, it is hypothesized that the
gene(s) in GI 148-1 which confer this resistance are likely dominant and donated by the
P. canescens parent. Disease reactions like that exhibited by GI 148-1 are typical of a
resistance that is simply inherited or race specific (Parlevliet, 1979). The dramatically
different reactions exhibited by triploid hybrid siblings GI 148-1 and GI 148-2 suggest
that the resistance gene(s) within GI 148-1 were not homozygous in the P. canescens
parent. Unfortunately the P. canescens parent used in the original cross in Germany has
not been maintained and is therefore unavailable to test this hypothesis (H. Schmidt, per.
communication). Due to its increased disease resistance, GI 148-1 is currently being used

in a backcross breeding program to confer resistance into sour cherry.

l7

CONCLUSIONS

Previous studies by Westwood et al. (1976) documented a spectrum of disease
reactions of MxM selections to B. jaapii ranging from highly resistant to extremely
susceptible. However, the two selections used in this study were susceptible to cherry
leaf spot and therefore will not be used as parental species for crosses with sour cherry.

Although P. serotina has been documented as susceptible to certain races of B.

jaapii, C. Iutescens Higgins (Higgins, 1914, Keitt, 1918; Heald, 1933), in this study the
P. serotina seedlings exhibited an immune response to cross infection of B. jaapii strains
isolated from P. cerasus leaves. In accordance with Keitt’s results (1918), the P. serotina
spp. capuli seedlings from Mexico and Ecuador are immune to infection from strains of
B. jaapii.

German hybrid, GI 148-1 exhibited resistance to infection by B. jaapii, in contrast
to its full sibling, GI 148-2 which was highly susceptible to disease. Identification of an
interspecific hybrid with increased disease resistance underscores the importance of

screening wild species and interspecific hybrids for increased disease resistance.

18

LITERATURE CITED

l9

LITERATURE CITED

Andersen, R.L. 1981. Cherry cultivar situation. Fruit Var. J. 35:83-92.

Cummins, J .N. 1979. Interspecific hybrids as rootstocks for cherries. Fruit Var. J.
33:85-89.

Davis, W.C., E. Wright, and C. Hartley. 1942. Diseases of forest-tree nursery stock.
Fed. Sec. Aganey Civ. Conserv. Corps For. Pub]. 9.

Eisensmith, SP. and AL. Jones. 1981. A model for detecting infection periods of
Coccomyces hiemalis on sour cherry. Phytopathology 71:728-732.

Eisensmith, SP. 1981. Infection model for timing fungicide applications to control
cherry leaf spot. Plant Disease 65:955-958.

Eisensmith, S.P., T.M. Sjulin, A.L. Jones and CE. Cress. 1982. Effects of leaf age and E
inoculum concentration on infection of sour cherry by Coccomyces hiemalis.
Phytopathology 72:574-577.

Garcia, SM. and AL. Jones. 1993. Influence of temperature on apothecial development
and ascospore discharge by Blumeriellajaapii. Plant Disease 77:776-799.

Heald, F .D. 1933. Manual of Plant Diseases. McGraw-Hill Book Co., Inc. New York.
pp. 551-562.

Higgins, BB. 1914. Contribution to the life history and physiology of C ylindrosporium
on stone fruits. Amer. J. Bot. 1:145-173.

Howell, GS. and SS. Stackhouse. 1973. The effect of defoliation time on acclimation
and dehardening in tart cherry (Prunus cerasus L.). J. Amer. Soc. Hort. Sci.
98:132-136.

lezzoni, A., H. Schmidt and A. Albertini. 1990. Cherries (Prunus) pp. Genetic Resources
of Temperate Fruit and Nut Crops (eds. JN Moore and J .R. Ballington) Acta Hort
290: 111-173.

Jones, AL. 1976. Diseases of Tree Fruits. North Central Regional Extension
Publication. 45: 33pp.

Jones, AL. and GR. Ehret. 1980. Resistance of Coccomyces hiemalis to benzimidazole
fungicides. Plant Disease 64:767-769.

20

Jones, A.L., G.R. Ehret, and SM. Garcia. 1993. Control of cherry leaf spot and powdery
mildew on sour cherry with alternate-site applications of fenarimol, mycolbutanil
and tebuconazole. Plant Disease 77:703-706.

Karsten, PA. 1884. Cylindrosporium padi Karst. (n. sp.). Mycol. Fenn. 16:159.

Keitt, G.W. 1918. Inoculation experiments with species of C occomyces from stone
fruits. J. Agric. Res. 13:539-569.

Keitt, G.W., E.C. Blodgett, E.E. Wilson and RD. Magic. 1937. The epidemiology and
control of cherry leaf spot. Univ. Wise. Agric. Expt. Sta. Res. Bull. 132. l77pp.

Olden, E.J. and N. Nybom. 1968. On the origin of Prunus cerasus L. Hereditas 59:327-
345.

Parlevliet, J .E. 1979. Components of resistance that reduce the rate of epidemic
development. Ann. Rev. Phytopathol. 17:203-222.

Popenoe, W. and A. Pachano. 1922. The capulin cherry. Journal of Heredity XIII
(2)250-62.

Popenoe, W. 1924. Hunting new fruits in Ecuador. Natural History, XXIV. Pp. 455-
466.

SAS Institute. 1989. SAS/STAT User’s Guide, Version 6. Fourth Ed. SAS Institute
Inc., Cary NC.

Schein, RD. 1964. Design, performance and use of a quantitative inoculator.
Phytopathology 54:509-5 13.

Schmidt H. and W. Gruppe 1988. Breeding dwarfing rootstocks for sweet cherry.
HortSci. 23(1):]12.

Sjulin, T.M., A.L. Jones and R.L. Andersen. 1989. Expression of partial resistance to
cherry leaf spot in cultivars of sweet, sour, duke and European ground cherry.

Plant Disease 73:56-61.

Stanosz, GR. 1992. Effect of cherry leaf spot on nursery black cherry seedlings and
potential benefits from control. Plant Disease 76:602-604.

Stairs, GR. and WT. Hauck. 1968. Reproductive cytology of black cherry (Prunus
serotina Ehrh). In: Proc 15‘'1 NE For Tree Improvement Conf, Morgantown,
West Virginia. pp. 42-53.

Von Arx, J .A., 1961. Uber Cylindrosporium padi. Phytopath. Z. 42:161-165.

21

Westwood, M.N., A.N. Roberts, and H0. Bjomstad. 1976. Comparison of mazzard,

mahaleb, and hybrid rootstocks for ‘Montmorency’ cherry (P. cerasus L.). J.
Amer. Soc. Hort. Sci. 101 :268-269.

22

CHAPTER TWO

GENETIC DIVERSITY WITHIN AND AMONG BLACK CHERRY

(PR UN US SEROTINA) FROM MICHIGAN, MEXICO AND ECUADOR

23

ABSTRACT

Black cherry (Prunus serotina Ehrh) is a common secondary forest species with a
wide endemic distribution, ranging from Nova Scotia into Mexico, Ecuador, and Peru.
Although heavily planted for its valued lumber within the United States, P. serotina is
essentially a wild species with small fruit (6-10 mm in diameter). In Mexico and
Ecuador, however, domesticants and feral populations of this species called “Capulin,”
have much larger (2-2.5 cm), edible fruit. No information is available concerning the
genetic diversity within P. serotina subsp. capulin, or between Capulin and P. serotina
subsp. serotina, the common black cherry of the US. Our objective was to investigate
the patterns of genetic diversity within and among black cherry germplasm collected from
Michigan, Central Mexico, and Ecuador. The following molecular marker systems were
informative: one chloroplast PCR product, two isozymes, and four sequence tagged sites
(STS) primer pairs from P. cerasus, P. avium, and P. persica. Three chloroplast
polymorphisms were identified with the most common fragment present in all three
geographic groups. All of the putative allozymes were identified in each P. serotina
population. STS markers identified the most genetic diversity. Mexican P. serotina
germplasm had the largest number of putative alleles, many of which were common to
the Michigan and Ecuadorian germplasm. Unique putative alleles were identified in the
Michigan and Ecuadorian germplasm, however, these two populations had no common
putative alleles that were not also present in the Mexican germplasm. This suggests that
the Michigan and Ecuadorian germplasm are divergent, most likely due to ecological and

geographic adaptation, selection and domestication events.

24

INTRODUCTION AND LITERATURE REVIEW

There are approximately 400 species within the genus Prunus, only twenty-five of
which are native to North America (Maynard et al., 1991). Among these species endemic
to the New World, the black cherry (P. serotina) is the only species commercially grown
for its high valued hardwood within the United States. P. serotina is commonly found as
a wild species in secondary forests (Maynard et al., 1991), thriving in recently disturbed
areas such as logging sites. Although P. serotina has been heavily cultivated and studied
by the forestry sector for decades, the small and non fleshy Northern P. serotina fruit (6- '
10 mm in diameter) has no commercial value (McVaugh, 1951). In Mexico and into the
Andean Highlands, however, domesticants and feral populations of this species called
“Capulin” have much larger edible fruit with large pits (Avg. 2-2.5 cm) (Popcnoe and
Pachano, 1922; Popenoe, 1924). It is generally accepted that the large fruited Capulin
resulted from domestication by native peoples in Central America (Popcnoe and Pachano,
1922)

Essential to the sour cherry breeding program at Michigan State University is the
acquisition of germplasm that might be used as donor species for disease resistance. P.
serotina was of particular interest because it is a tetraploid species (x = 8, 2n = 32) (Stairs
and Hauck, 1968) like sour cherry (Olden and Nybom, 1968) and therefore may have the
ability to produce fertile progeny when hybridized with sour cherry. For scion cherry
breeding, the Capulin cherry may be the most useful P. serotina subspecies parent

because of its large fruit size.

25

Termed one of the ‘lost crops of the Inca’s,’ no information is available
concerning the genetic diversity within Capulin, or between P. serotina subsp. capulin
and the black cherry of the United States, P. serotina subsp. serotina. Molecular data
which describes the genetic relationships within and among P. serotina populations
would aid in the selection of parental P. serotina material, as well as reveal insights into
its evolutionary history. Our goal is to characterize the natural genetic diversity within P.
serotina by sampling selections from Michigan, Ecuador and Central Mexico using
several molecular marker systems.

Background

P. serotina has a large endemic distribution ranging from Nova Scotia down the E
eastern US. coast, with disj unct populations also in west Texas, southern Arizona and
New Mexico. This species is also endemic to Central Mexico and into Ecuador and Peru
(McVaugh, 1951). It is generally accepted that the Spaniards introduced P. serotina from
Mexico or Central America into the Andean region during Colonial times (Popcnoe et al.,
1989). Although not formally cultivated, the Capulin black cherry is domesticated, ofien
harvested and sold in fresh fruit markets in the Andean region. P. serotina was
subsequently introduced into Europe from the US. in the 17‘h century initially as
decoration in gardens and parks. Due to its prolific characteristics in these areas, P.
serotina is often referred to as a forest pest rather than a valued ornamental species today
in Europe (van den Tweel and Eijsackers, 1987).

P. serotina of the US. and Canada differ from those in Mexico and Ecuador only
by those horticultural traits that are easily manipulated by selective cultivation (Popcnoe

and Pachano, 1922). Mexican and Ecuadorian P. serotina trees usually grow as multiple-

26

stemmed trees of approximately 35 feet in height. In contrast, forestry planted trees in the
US. are selected for and grown as straight, single trunked trees with heights of 120 feet
(Maynard et al., 1991). The following descriptions of P. serotina are taken from
McVaugh (1951):

“The species as a whole is characterized as having alternate and simple oblong to

lanceolate leaves with fine double toothed serrations. The bark has the

characteristic horizontal lenticles of cherry species. The wood of black cherry is a

valued hardwood in the US. commonly used to make prized home furnishings

and interior paneling. Flowers of the black cherry are borne on loosely clustered
racemes with approximately 20-30 flowers per inflorescence. When ripe, fruit are
dark red to black. The name ‘Capuli or Capulin’ is used only to designate the
larger fruited varieties.”

Typically, the fruit of P. serotina in the US. and Canada averages 6-10 mm in
diameter (McVaugh, 1951). Flesh of the black cherries are bitter in taste and typically
surround large pits. Regardless, the fruit is consumed by many bird species and has been
used in folk medicines and to flavor liquors. The larger, edible Capulins of Central
Mexico Mexico have long been incorporated into local cultures. Ranging from 2 cm to
the most famous “Gonzales Capulin” in Catiglata, Ecuador measuring 3.5 cm in diameter
(Popcnoe and Pachano, 1922) P. serotina continue to be domesticated. The largest of the
Capulins in Ecuador are harvested and found frequently in fresh fruit and vegetable
markets (Popenoe, 1924).

In Central Mexico, P. serotina is a common unmarricured species in home
gardens and grows as a predominant species along roadsides and fence lines surrounding
a variety of orchards within the mountains northeast of Mexico City (Appendix A).

Although there is no formal cultivation, selection for the Capulin cherry is apparent by its

overwhelming presence in these selected areas, protected and cherished by local people.

27

While many people of Mexico eat large fruited Capulins as fresh fruit, the smallest and
darkest cherries are often used to make a brandy wine. The pits of the largest fruits are
toasted and eaten like pistachios (S. Perez, Per. Comm., 1996). In some areas, the fruit is
said to have a medicinal use as a tonic to relieve coughs (Appendix A).

Previous Research

Most formal research conducted on P. serotina has been through the forestry
sector. Evaluation and phenotypic selection of seedlings for superior timber qualities and
geographic distribution of naturally occurring P. serotina have dominated the literature
(Pitcher and Dom, 1972; Genys and Cech, 1974). Tissue culture manipulation as well as
proper handling of seed for maximum germination have also been important research
areas (Caponetti et al., 1971; Dills and Braharn, 1988; Tricoli et al., 1985; Dradi and
Biondi, 1991; Maynard et al., 1991; Forbes, 1972; Huntzinger, 1968). Little information
is available regarding the genetic diversity in P. serotina.

Since no information is available regarding the use of molecular markers in P.
serotina, molecular markers were chosen for this diversity study that had been shown to
be polymorphic in the horticulturally important cherry species (P. avium and P. cerasus).
Both chloroplast and nuclear markers were selected due to their different modes of
inheritance and rate of mutation. Chloroplast DNA (chNA) is maternally inherited in
cherry (Brettin, 1999) and it is especially useful for phylogenetic studies due to its high
degree of base sequence conservation (Curtis and Clegg, 1984; Palmer, 1987). Although
chNA is highly conserved within a species, non coding regions within the chloroplast
genome have higher rates of mutation, including insertions/deletions. Tablerlet (1991)

designed conserved primer sets in conserved regions flanking non-coding regions of the

28

 

chloroplast genome. Brettin et a1 (1999) used one of these sets, the ‘AB’ primer set, to
amplify one of these variable chloroplast regions and search for polymorphisms in sour
cherry. Within sour cherry seven length polymorphisms were found for the ‘AB’
fragment. To better define the insertion/deletion events resulting in these seven
polymorphisms, approximately 300 bp of sequence was obtained for these ‘AB’
fragments (Brettin et al, 1999). The first 300 bp of sequence revealed two
polymorphisms which differed for two insertion/deletions, one of 8 bp and another of 6
bp. Primers flanking these two insertions/deletions were designed and termed the nested
AB primers, [AB], to facilitate the rapid screening of sour cherry germplasm of the 14
base pair length polymorphism. This nested chNA primer pair was chosen for use in
this P. serotina study.

Two nuclear marker types were chosen; isozymes (enzymes with specific
catalytic activity) and sequence tagged sites (STSs, i.e. PCR amplified fragments where
the primers are designed from available sequence data). Both marker types are
codominant which permits the identification of the heterozygous class. Although
isozymes exhibit limited levels of polymorphism compared to other marker systems, they
were included in this analysis since we have previous information on them and the data
obtained represent actual plant proteins.

Isozymes have been used to study genetic diversity and polyploid inheritance in
sour cherry (Beaver and lezzoni, 1993). Two of the more polymorphic isozyme systems
with inheritance data available were chosen for our study, 6-PGD and PGI. Both isozyme
systems are dimeric and extensively studied in Prunus (Byme and Littleton, 1988; Parfitt

et a1, 1985; Byme, 1989, and Chaparro et al., 1987).

29

Microsatellites or simple sequence repeats (SSRs) are a group of sequence tagged
sites (STSs) where primers of specific sequences are designed to flank hypervariable
regions of di-, tri- or tetra-nucleotide repeats (Staub and Serquen, 1996; Queller et al.,
1993). Amplifications of these specific non-coding regions is performed by PCR. These
hypervariable non-coding regions of the nuclear genome are often highly polymorphic
within species, producing fragment length patterns which can frequently identify specific
progeny. At the time of this study, a limited number of SSR primer pairs were available
from sweet cherry (G. King, pers. com.) and sour cherry (A. lezzoni, pers. com.) The
other available STS primer pairs were designed from cloned amplified fragment length
polymorphism (AF LP) fragments in peach (A. Abbott, pers. comm.)

Objective

Our objective is to investigate the genetic relatedness of P. serotina selections
from Michigan, Ecuador and Central Mexico using several molecular marker systems and
provide baseline data from which future in depth studies of the evolution and

domestication of P. serotina and its relationship with other Prunus species could begin.

30

MATERIALS AND METHODS

Plant Material

P. serotina seedlings used in this analysis were from three sources: Mexico,
Ecuador and Michigan (Table 3). Accessions from Central Mexico were collected as
open-pollinated seed from under Capulin trees August 12-17, 1996 (Appendix A). Open-
pollinated seed was collected from twelve trees termed 12 collection ‘families’ (RG, P1,
PH, TI, P3, P2, ARG, U2, U3, J 1, J2, and J3). Three of these families (RG, TI and J3)
were collected by collaborators of Sr. Perez prior to our arrival in Central Mexico
(Appendix A). Ten Ecuadorian accessions were obtained from the Ecuadorian
Germplasm Institute as open-pollinated seed presumably collected from five trees termed
five collection ‘families’ (Ecu A 1-3, E 1, D 1-2, F 1-2, H 1-2) (Appendix B) [c/o Dr.
Raul Castillo, Departamento Nacional de Recursos F itogeneticos y Biotecholigia, Casilla
17-01-340, Quito, Ecuador]. Uneven progeny numbers from these five Ecuadorian
families resulted from poor seed germination. Ten P. serotina subsp. serotina plants
were obtained as young seedlings from the MSU Department of Forestry Extension
Service. These seedlings were collected as bulk seed from the P. serotina collection at
the Kellogg Biological Station. Selections of P. avium (Emperor Francis), P. cerasus
seedlings (‘Montmorency,’ Erdi Boterrno, Csengodi, and Rheinische Schattenmorelle),
and P. cerasus x P. canescens hybrids (GI 148-1 and GI 148-2) were used as reference
selections in the isozyme analysis. Plant material used for chloroplast fragment

amplification, isozyme analysis, and STS fragment amplification are presented in Table 3.

31

CHLOROPLAST MARKER SYSTEM
DNA Isolation and Quantification

Procedures for extraction and quantification of total DNA from P. serotina
seedlings were modified from Stockinger et al. (1996). After adding 10 ml of
chloroform;isoamyl alcohol 24: 1, each sample was homogenized by gentle shaking for at
least five minutes to separate and extract proteins from the DNA. When pellets failed to
dissolve in T10N700Eo,5, centrifugation time was decreased to 16 minutes and pellets were
dissolved at 60°C after incubation at 4C overnight. For all samples, young leaves were
harvested from seedlings and kept at -80°C overnight and subsequently lyophilized for
48 - 72 hours. Optimally, extraction yielded between 500ng - IOOOng/ul DNA per sample.
Dilutions of 50 ng/ul were made and used as working stock samples for each accession.
Sample Amplification and Fragment Resolution

For each 25 ul PCR reaction, the following reagents were used: 15.3 7ul
autoclaved ddH20, 2.5ul 10x PCR buffer, 1.0ul 25 mM MgC12, 2.0u1 10x dNTPs, 1.0ul
forward primer, 1.0111 reverse primer, 1.0ul of 50ng/ul diluted DNA template and 0.13 111
Taq polymerase. PCR parameters for the ‘AB’ amplification are listed in Table 4. Each
of the 24 samples in total were amplified using the ‘AB’ primers from the chloroplast
variable non-coding region (Tablerlet, 1991). PCR products from this reaction were
diluted 1:100. Successive PCR reactions using the nested primer pair, [AB], used the
diluted PCR product as template. The same PCR program was followed for the ‘AB’ and
nested [AB] amplification (Table 4). Visualization of amplified products was resolved on

6% polyacrylamide gels run on 80v for 2 hours and stained using the Silver SequenceTM

32

staining system (Promega, Madison, WI). Amplified fragments were scored against a
10bp ladder (Gibco, cat. #10821-015, Fredrick, MD).

NUCLEAR MARKER SYSTEMS

Isozyme Analysis

Starch gel electrophoresis was performed on protein extracts obtained from P.
serotina seedlings and reference plants. Young leaves and eltiolated buds were collected
and stored in eppendorf tubes at 4°C prior to enzyme extraction. All material was
macerated using cooled mortar and pestles in a walk in cooler to ensure temperature
stability and reduction of enzyme activity. Isozyme extraction procedures follow the
procedures of Krebs and Hancock (1989) with slight modification. No nylon screens
were used during the extraction process. In addition, extraction buffer for sample
maceration was maintained at pH 7.5 instead of pH 8.0.

Two isozyme systems representing two loci each, 6-phosphogluconate
dehydrogenase (6-PGD) and Phosphoglucose isomerase (PGI) were resolved on 12%
potato starch gels with approximately Six and a half hours of electrical current. 6-PGD
was resolved on a morpholine-citrate pH 6.1 gel (Clayton and Tretiak, 1972) while PGI
resolved best on a tris-citrate/lithium-borate pH 8.3 gel (Scandalios, 1969). For each gel
system, an electrical current of approximately 50mA was maintained until wicks were
removed after 30 minutes. Electrophoresis was continued for six hours without
exceeding 300 V. Both stain recipes for PGI and 6-PGD were prepared as directed in

Arulsekar and Parfitt (1986) at a volume of 50 ml per gel slice.

33

The gels were scored following the general procedures of Beaver et al. (1995).
Band mobility and allelic designations of P. serotina isozyme samples were compared to
out group controls as reported by Beaver et al. (1995).
SEQUENCE TAGGED SITE AMPLIFICATION
DNA Isolation and Quantification

Procedures for extraction and quantification of total DNA from black cherry
seedlings were as reported above.
Sample Amplification and Fragment Resolution

In total, eight Sequence Tagged Sites (STSs) primer pairs were used to identify
informative markers. Optimization of PCR conditions for each primer pair was done by ‘
using a temperature gradient on a Robocycler (Stratagene, La Jolla, CA). PCR conditions
for each primer pair and STS are listed in Table 5. For each 25 u] PCR reaction, the
following reagents were used: 15.37ul autoclaved ddH20, 2.5 ul 10x PCR buffer, 1.0ul 25
mM MgC12, 2.0ul 10x dNTPs, 1.0ul forward primer, 1.0ul reverse primer, 10111 of
50ng/ul diluted DNA template and 0.13 [.11 Taq polymerase. Three SSR amplifications
were attempted using one primer pair from sour cherry, GA34, and primer pairs from
sweet cherry, PSl2A02 and PSO8E08 (Appendix C). Primer pairs B3D5, B10B9, B6B],
BlOH3, and B4G3 of peach origin (Appendix C) were also used.

Visualization of amplified products was resolved on 6% polyacrylamide gels run
on 80v for 2 hours and stained using the Silver Sequence“ staining system (Promega,
Madison, WI). Fragment length patterns were scored against a 10 bp ladder (Gibco, cat.

#10821 -01 5, Fredrick, MD).

34

RESULTS '

Chloroplast Amplification

Amplification of the chloroplast nested [AB] fragment between the non-coding
region between the trnT and trnL genes in Prunus was successful in P. serotina,
indicating that this region within the chloroplast genome is conserved across cherry
species. As expected, only one fragment was amplified in each reaction (Table 5, Figure
3). Amplification of the chloroplast non-coding region identified fragments of three
different lengths, 274 bp, 280 bp, and 250 bp. All Michigan selections (MI 1-10),
Mexican selections (PH, TI, P2 and P3) and Ecuador P. serotina selections (Ecu A, E, F, '
and H) had the 274 bp fragment (Table 5, Figure 3). Two of the three samples from
Mexican accession RG had one fragment of 250 bp. The third sample from the RG
collection family exhibited a fragment of 280 bp. One Ecuadorian accession, Ben D, also
had the 280 bp fragment.

The 280 bp fragment exhibited by the Ecuadorian accession Ben D is also not
unexpected because Ecuadorian accessions were probably derived from multiple maternal
material as well (Appendix B). Morphologically, the RG and Ben D families were
indistinguishable from other collection families and subsequent assays with isozymes and
STSs show no further deviation of these accessions from the rest of the Capulin gene
pool. Therefore, chNA divergence is most likely due to collection of open pollinated
seed from multiple maternal trees. Resolution at 280 bp for both Ben D and RG supports
the theory that Mexican and Ecuadorian P. serotina still share much of their genetic

background with one another, despite geographic isolation between the two.

35

Amplification of the 274 bp fragment suggests that collection families from Michigan,
Mexico and Ecuador share a common gene pool, despite the limited geographic sampling
from the Northern P. serotina. Of course, to get a more accurate View of the true
diversity within this species, a larger sampling of Northern black cherry from various
geographic locations is necessary.

Isozyme Analysis

Two loci were resolved well and exhibited good activity for phosphoglutose
isomerase, Pgi-l and Pgi-2 (Figure 4). The Pgi-l locus was monomorphic for the
putative allele Pgi-l '05 for all P. serotina progeny tested. Pgi-2 locus exhibited three
different alleles in the P. serotina selections tested, Pgi-2IOO, Pgi-280 , and Pgi-270 . Pgi- '
1'05 and Pgi-280 were found in both the P. serotina selections as well as the reference
selections P. avium cv. Emperor Francis, P. cerasus cvs. Csengodi, ‘Montmorency’ and
Rheinische Schattenmorelle. For Pgi-2, the P. serotina selections either exhibited one or
two putative alleles per locus (Figure 5): (1) Pgi-Z'OO and Pgi-280 and the associated
heterodimer (Pattern 1), (2) Pgi-2loo and Pgi-270 and the associated heterodimer (Pattern
2) or (3) only the Pgi-2100 allele (Pattern 3). All three allelic patterns encoded at the Pgi-2
locus were exhibited by selections of P. serotina from all three of the geographic
collections, Michigan, Mexico and Ecuador (Figures 4 and 5).

Two loci for 6-phosphoglutonate dehydrogenase, 6-Pgd-1 and 6-Pgd-2 resolved
clearly in Mexican and Ecuadorian P. serotina progeny (Figures 6 and 7). Unfortunately,
Michigan progeny assayed for 6-PGD repeatedly failed to resolve bands with clarity and
therefore scoring the bands was not possible. For the P. serotina selections assayed, two

putative alleles at each locus were identified, 6-Pgd-l '05 and 6-Pgd-194, and 6-Pgd-288

36

and 6-Pgd-266 (Figure 7). None of these putative alleles were found to be in common
with reference P. cerasus, P. avium, and P. cerasus x P. canescens selections. Banding
patterns of 6-PGD could differentiate enzymes extracted from P. serotina from those
extracted from sweet and sour cherry. All P. serotina progeny tested exhibited the 6-Pgd-
l105 and 6-PgaI-266 putative alleles. However, some selections were heterozygous at both
loci (Pattern 1, Figure 7), heterozygous at 6-Pgd-l (Pattern 2, Figure 7) or heterozygous at
6-Pgd-2 (Pattern 3, Figure 7). Progeny from both Mexico and Ecuador exhibited all three
patterns for 6-PGD. Diagnostic alleles were only found in one non-P. serotina reference
group. In this case, GI 148-1 can be distinguished from its half sibling, GI 148-2 (Figure
7).
STS Analysis

A summary of informative and non-informative primer pairs from sweet and sour
cherry and peach on P. serotina selections can be found in Table 6. Of the eight sequence
tagged site (STS) marker systems attempted, two primer sets failed to amplify fragments
in P. serotina. Both peach derived primer sets B3D5 and BlOB9 repeatedly failed to
yield fragments. Two primer pairs, one derived from peach (B4G3) and one derived from
sweet cherry (PSO8E08) exhibited monomorphic banding patterns across P. serotina from
Michigan, Mexico and Ecuador. Primer set B4G3 yielded a 166 bp fragment and
PSO8E08 exhibited a 138 bp fragment for all P. serotina selections. Due to the lack of
diversity in fragment sizes for these two primer pairs, they were deemed uninformative.

Four of the eight primer pairs tested were found to identify polymorphisms, one

derived from sweet cherry (P812A02), one derived from sour cherry (GA 34) and two

derived from peach (B6B1 and BlOH3). GA34 and P812A02 amplify SSR containing

37

sequences, while B6B1 and BlOH3 amplify AF LP-identified regions of the genome.
Together, these four primer pairs resolved 54 putative alleles for the 66 P. serotina
selections assayed (Table 6).
SSR Amplification with Sweet Cherry Derived Primers

Amplification with sweet and sour cherry derived primers was successful. These
hypervariable regions (GA34 and PSl2A02) are conserved between P. avium L. and P.
serotina. Sour cherry derived primer pair GA34 amplified a total of fourteen fragments
ranging from 140-174 bp in length (Appendix C and Table 6). A maximum of two
fragments were amplified per individual sampled. Of the fourteen putative alleles, one
(140 bp) was specific to Ecuador, two (150 and 160 bp) were specific to Mexico, and two
fragments were identified only in Michigan P. serotina selections (170 and 174 bp)
(Figure 10 and 11). Two fragments (146 and 162 bp) were shared between Michigan and
Mexico gene pools only. Fragments 144, 154, 158, 160, 164, and 168 bp were shared
among the Mexican and Ecuadorian accessions only. One fragment, 148 bp, was found
in all three gene pools. No putative alleles were shared among the Michigan and Ecuador
germplasm without also being present within the Mexican gene pool (Figures 8 and 9).

Primer pair P812A02 was also highly informative, producing twelve putative
alleles with a maximum of four fragments amplified per accession (Table 6). For this
primer set, only four fragments were specific to any one particular gene pool: 164, 172,
176, and 178 bp all identified only Michigan selections. Four fragments were shared
between Mexico and Michigan (150, 156, 162, and 168 bp) while three fragments were
shared between Mexico and Ecuador (154, 160, and 170 bp) (Figures 8 and 10). Only

fragment 148 bp was shared among all three gene pools. Again, no putative alleles were

38

shared between Ecuador and Michigan selections without also being present within the
Mexico gene pool.
STS Amplification with Peach Derived Primers

Peach primer set B6B] resolved the most putative alleles with nineteen in total,
ranging from 170 to 230 bp in length (Table 6, Figures 11 and 12). A maximum of four
fragments were amplified for all P. serotina progeny assayed. None of the fragments
amplified were specific to Michigan or Ecuador gene pools. However, five alleles were
unique to Mexican accessions (176, 196, 214, 226, and 228 bp). Two alleles were shared
between Mexico and Ecuador P. serotina (202 and 222 bp), while five alleles (186, 188,
192, 210 and 230 bp) were shared between Mexico and Michigan P. serotina selections. ’
All together, seven putative alleles were shared among all three gene pools: 170, 180,
190, 200, 216, 220, and 224 bp. Once again, no putative alleles were exclusive to the
Michigan and Ecuador selections without also being present within the Mexican P.
serotina gene pool.

Peach derived primer set BlOH3 also successfully amplified fragments from all P.
serotina selections from Michigan, Mexico and Ecuador. Nine putative alleles were
resolved using this primer set, with a maximum of two fragments per sample (Table 6,
Figures 11 and 13). Using this primer set, three putative alleles identified specific gene
pools: 130 bp (Michigan), 150 bp (Mexico) and 132 bp (Ecuador). No fragments were
shared among all three gene pools. Four alleles were shared between Mexican and
Ecuador P. serotina selections (138, 140, 142, and 144 bp). Only two fragments were
exclusively shared between Mexico and Michigan P. serotina selection, 146 and 152 bp.

As with the previous STS markers, no putative alleles were found to be exclusively

39

shared between the Michigan and Ecuador P. serotina selections without also being
present within the Mexican germplasm.

A total of nine STS fragments were shared among all three geographically
distinct groups. Thirteen fragments were shared among the Michigan and Mexican
groups, while fifteen were shared exclusively between the Mexican and Ecuadorian
groups. Of all the informative STS fragments, none of the putative alleles were shared
between the Michigan and Ecuadorian P. serotina families without also being present
within the Mexican families. Each geographically distinct group exhibited putative
alleles which were diagnostic to their group (7 Michigan, 8 Mexican, and 2 Ecuadorian
fragments). Although all three geographically distinct groups of P. serotina share a large '
number of putative alleles, amplified fragments which identified progeny to Mexican,
Michigan, or Ecuadorian groupings were common in the STS markers. However, none of
the putative alleles assayed could be used to identify progeny trees to their respective

collection families such as RG or Ben E.

40

Table 3. Number of open-pollinated P. serotina seedlings per family assayed for
chNA fragments, 6-PGD, PGI, and sequence tagged sites (STSs).

 

P. serotina
Family chNA
Origin Family Fragment 6-PGD P
MEXICO RG 3
PH
P1
P2
P3
TI
ARG
U2
U3
11
J3
ECUADOR Ecu A
Ecu D
Ecu E
Ecu F
Ecu H
MICHIGAN MI
Beal

I STSs

 

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I
immu—Nwmmmmmmmmmmm
NNHNwmmmmmmmmmmmn
I

fl
0
t-ilJrNNt-‘NUJI

Hugged—aha.

 

41

 

35—m—

 

 

 

 

 

 

58 m \omn mm 58 mg \omh Es: >8 58 _ \0g 58 m \ovm mann—
Nc<§mm
58 m \omn mm 58 m; \omn 58 fl \owm EE _ ES 58 m \ovo MU:—
mic—m
35m
58 m \omh mm £8 m; \omn ES _ >8 58 _ \ovm 58 m \ovm 2.”:ch
v35
:3
58 m \omh mm 5:: \omh ES _ \oom ES _ \ovo 58 m \ovo m<
oEw—KQEoH no.9.m0 we oar—Each mun—FEE“; air—knack. earth—nob
ucfiuounm LEE: Z comm—Baum ans—«2:3. 9.5.2.09 9:525: a: he.— 3.5.:—
eio as. 586 2&0 2m

 

.uocmsom can docs—2 .cmeOME Bot 653.8%. .k E Ammhmv mozm nowwfi 8:258 8305: can
5&2 wemvooéoe awe—mecoEo m @295 9 3m: :8 cos—ta some now £599.00 don BNEEEO .v 03m...

42

Table 5. Fragments amplified from successive PCR reactions on a chloroplast
non-coding region [AB] from P. serotina selections from Michigan,
Mexico and Ecuador.

 

No. No. Bands No. Bands
Origin of Selection Samples Amplified Resolved at
Selections ID Assayed Per Lane 250bp 274bp 280bp

 

p—n
N

MEXICO RG 3
PH
TI
P2
P3
ECUADOR Ecu A
Ecu D
Ecu E
Ecu F
Ecu H
MICHIGAN Ml
Beal

D—‘Wr—au—Ir—ou—Ir—IUJUJUJDJ

p—au—ar—or—au—‘u—‘r—au—nu—av—ar—a
I I

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I

 

43

 

 

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com am 2 M QOm
o o com mm 3. M QOm :03?—
ooo mm 2 M womgmm
_ M cow mm 2 "— wommwomm
com mm 2 M NOAH—mm
S v com em 2 M NO<N~mm
ace 2 2 M «3.6 Cram—=0
E N coo cm 7... r.— vm<O hmmgm
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aoam .8..— m—Eam magm— =£28EEE< and 0:32 no.5...— .__a.— 3.5...—
.e2 .39.. 52 835.32

 

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Eamon use Pogo 3026 GEM @963 E3 SEEM gums—SETS: Ea gums—LAME Mo Egm .0 03m...

44

MI MEX

 

280—9'

274 —>

250 —>

 

 

 

Figure 3. Amplified non-coding regions of P. serotina chloroplast genome
resolved on 6% polyacrylamide gels and stained with Silver Sequence”
Stain. Gels are oriented with loading origin at the top of each photograph.

45

GI 148-l
GI 148-2

H

 

Figure 4. Pgi-l and Pgi-2 loci patterns in P. serotina samples from Michigan
Mexico, and Ecuador. Gels are oriented with the sample loading
origin at the bottom of each photograph. Selected loci assigned alleles
and out groups GI 148-1 and GI 148-2 are labeled with arrows.

46

 

 

 

Assigned Banding Patterns
Band Allele
Locus Size 1 II 1 2 3 4 5
Pgi-l 110 B .
105 a A O C O .
Pgi-2 100 a A O O O
90 O
80 b B O O O O
70 c O O
60 D .

 

 

Figure 5. Zymogram representing patterns exhibited by P. serotina selections from
Michigan, Mexico and Ecuador and out group controls assayed for
Phosphoglucose isomerase (PGI). Alleles I represent putative alleles for P.
serotina. Alleles 11 represent putative alleles for out group controls. Pattern 1:
Michigan (MI), Mexican (U2, ARG, P3, RG, U3, J 1, P1, TI, PH, and J3), and
Ecuadorian (Ecu A-H) P. serotina and EB (Erdi Botermo). Pattern 2: Michigan
(MI), Mexican (P2, J 1, P1, TI and U3), and Ecuadorian P. serotina (Ecu A-H).
Pattern 3: Michigan (MI), Mexican (U3 and RG) and Ecuadorian P. serotina (Ecu
A-H). Pattern 4: Emperor Francis (EF), Csengodi, Montmorency, and Rheinische
Schattenmorelle (RS). Pattern 5 was only exhibited by GI 148-1 and GI 148-2.

47

105 _.
94 "

 

 

Figure 6. 6-Pgd-1 and 6-Pgd-2 loci patterns in P. serotina samples from
Michigan Mexico, and Ecuador. Gels are oriented with the sample
loading origin at the bottom of each photograph. Selected loci
assigned alleles are labeled with arrows.

48

 

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A:-< somv grown—Sm 28 CM new mac 5&on ”m :6th .mcommmooom Am-< somv camcowmsom new
9 m 98 mm .OM< .7: 58:82 ”N :5th $56303 3533. .m Amaze sum: auto—oesom can
5 Ba .3 .E .2 .92 .B .3 .E .E .2 .E 5832 ; 5.58 mega sates 92w
So no.“ 33% 2683 Beanie M mo_o__< 62.23%. .m com 83:.“ 9553 €3,052 _ mo_o=<
.AQOMOV omwcowohinow Bassoon—wofimonaé com 3.3.0.3 £888 95cm So 28 833m new
02on 80¢ 32828 annexes. .& .3 3:92.38 mEozmm weaves mass—OmEQB ESonAN .5 0.5me

 

 

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e m e m N _ = _ SE 2.8..
22:. 2:5
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2:03am wE—Eam Illr

 

 

 

49

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.33 couaouzasa 88m was—32 22338 32.238. .& 835on was oomon .SwwEog .8.“ “no—23 933:“—
.Euno $93 98 50m Set Ecu—HS: mhm clung—£5 mE>_om2 20» 02.5108on $0 mo Eng—woaonm .w oBmE

 

 

 

 

50

MEXICO

 

MICHIGAN ECUADOR

Figure 9. Putative alleles identified by STS GA34 for Michigan, Mexico and
Ecuador P. serotina selections. Putative allele numbers correspond to
amplified band sizes (bp).

51

MEXICO

164, 172
176, 178

 

MICHIGAN ECUADOR

Figure 10. Putative alleles identified by STS PSlZAOZ for Michigan, Mexico
and Ecuador P. serotina selections. Putative allele numbers correspond to
amplified band sizes (bp).

52

 

.'7- ' ‘

.._. fl Iii Q... ‘* as aw in”
.3" 6 - Sufi“; '1
“aw .. *4“ 9'

. ... "5’

‘5 _ ._ * nut

paw-

 

 

 

 

 

Figure 11. Photographs of informative STS marker systems from peach resolved on 6%
polyacrylamide gels. Putative alleles for Michigan, Mexico and Ecuador P.
serotina accessions resulting from amplification with primer pair B6B1 (A) and
BlOH3 (B&C).

53

MEXICO

      
 
  

214 228 226 196
176

186 188
192 210
230

  

  
 
 
   
   

   
  

170
180 190
200 216
220 224

MICHIGAN ECUADOR

Figure 12. Putative alleles identified by STS B6Bl for Michigan, Mexico and

Ecuador P. serotina selections. Putative allele numbers correspond to
amplified band sizes (bp).

54

 

MEXICO

 

138, 140

MICHIGAN ECUADOR

Figure 13. Putative alleles identified by STS BlOH3 for Michigan, Mexico and
Ecuador P. serotina selections. Putative allele numbers correspond to
amplified band sizes (bp).

55

DISCUSSION

Evidence of shared gene pool among the Mexican, Michigan, and Ecuadorian
germplasm is evident, based upon the similar patterns and putative alleles identified by
the chloroplast and nuclear markers, respectively. Even with limited sampling from
Ecuador and Michigan, there is evidence that these geographically distinct populations,
while sharing a common gene pool, exhibit divergence which likely has resulted from
geographic and ecological adaptation, selection pressures and domestication events in
Central America.

Since chNA is maternally inherited in Prunus, it was expected that all the open-
pollinated seedlings from one family would have the same chloroplast fragment.
However, seedlings from the Mexican family RG exhibited different fragment sizes (250
bp and 280 bp). Two different fragment sizes exhibited by Mexican family RG can be
explained by field collection procedures. For the RG family, open-pollinated seed was
collected prior to my arrival in Mexico (Appendix A). Until chNA results, it was
assumed that the open-pollinated seed was collected from only one maternal parent tree.
However, the chNA data suggests that multiple maternal parents were sampled for this
family.

The 280 bp fragment exhibited by the Ecuadorian accession, Ecu D, may have
resulted because Ecuadorian accessions were probably also derived from multiple
maternal parents (Appendix B). Morphologically, the RG and Ecu D families were
indistinguishable from other collection families and subsequent assay with isozymes and

STSs show no further deviation of these accessions from the rest of the Capulin gene

56

pool. Therefore, chNA divergence is most likely due to collection of open-pollinated
seed form multiple maternal trees and not due to sampling of other Prunus species.

Nested [AB] chloroplast primers designed based upon P. cerasus sequence
identified three different length fragments in P. serotina. The most common fragment
had a length of 274 bp. This may be the most “ancestral fragment,” and the other
polymorphisms of 250 bp and 280 bp may have resulted from a 24 bp deletion even(s)
and a 6 bp addition event(s), respectively. Such insertion and deletion polymorphisms
have been reported to account for a substantial fraction of intraspecific chNA variation
(Zurawski and Clegg, 1987). Interestingly, two of the three fragments amplified in P.
serotina (274 and 250 bp) are similar in length to those identified in French sweet cherry
selection (FPA 276bp) and in sweet, ground and sour cherry selections (249 bp)
respectively (Brettin, et al., 1999). Sequence data for the P. serotina fragments would be
necessary for comparison with sour cherry.

The common 280 bp chloroplast fragment for both Ecu D and RG supports the
theory that Mexican and Ecuadorian P. serotina still share a similar genetic background
wit one another, despite their geographic isolation. Amplification of the 274 bp fragment
in the families from Michigan, Mexico and Ecuador suggest that germplasm from these
regions share a common gene pool. Of course, to get a more accurate view of the true
diversity within this species, a larger sampling of Northern P. serotina from various
geographic and ecological locations is necessary.

Nuclear markers also identified unique putative alleles among the collection
families form Michigan, Mexico and Ecuador. As expected, isozymes exhibited less

diversity than the non-coding hypervariable regions of the nuclear genome amplified by

S7

the PCR based STS marker systems. Putative isozyme alleles identified were conserved
across all three geographical P. serotina groupings. Isozyme data supports the theory that
P. serotina from Michigan, Mexico and Ecuador remain genetically similar on the protein
level, despite their morphological differences.

Successful amplification of nuclear regions using primer sets derived from
distantly related Prunus species has been demonstrated indicating that these primer
sequences are conserved. However, not all primer sets were successful. For example,
two of the six peach primer sets failed to amplify and fragment, two others produced only
monomorphic fragments, and the remaining primer sets, B6B] and BlOH3 were the only
informative primer pairs. None the less, our results suggest that useful P. serotina STS I
primer pairs can be selected from those available for peach and cherry.

STS marker systems were the most informative for this diversity analysis due to
the relatively large number of putative alleles identified among the P. serotina selections.
This was expected since non-coding DNA regions, particularly short repeat regions, have
a higher occurrence of insertion and deletion events. Only STS markers were unique for
the different geographic groupings. No more than four putative alleles per progeny
assayed were amplified by the STS markers. A maximum of four alleles would be
expected since P. serotina is a tetraploid and four alleles would represent the maximum

number of alleles at two duplicate loci.

58

CONCLUSIONS

This is a preliminary study of the genetic diversity found within P. serotina from
Michigan, Mexico and Ecuador. Its primary application is to derive baseline molecular
data and identify primer sets that may be useful for more extensive and comprehensive
inquiries in the firture. The conclusions made are based upon an extremely limited
sample sizes taken from specific regions of Michigan, Mexico, and Ecuador. No broad-
based conclusions should be made from this data without further investigation.

Of the marker systems assayed in this study, the STS nuclear markers provided
the most diversity of putative alleles within the P. serotina selections. The Mexican P. I
serotina germplasm was the most diverse geographical grouping, sharing many common
alleles with the Michigan and Ecuadorian germplasm. Despite the similar gene pools,
however, there does appear to be some genetic divergence between Michigan and
Ecuadorian germplasm, most likely due to ecological and geographic adaptation,
selection and domestication events. This is supported by chloroplast and STS marker
data. Although this diversity study is preliminary due to the small amount of germplasm,
the data does suggest that he molecular markers identified would provide a useful starting

point to investigate the evolution and domestication of the large fruited Capulin cherries.

59

LITERATURE CITED

6O

LITERATURE CITED

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6]

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63

APPENDICES

64

APPENDIX A

COLLECTION INFORMATION FOR MEXICAN P. SEROTINA SELECTIONS

6S

The following tables are a summary of information collected on P. serotina subsp.
capulin species collected in Mexico during August 12-17, 1996. Collection information,
physical data, as well as cultural notes are included. Twelve accessions were collected as
open-pollinated seed from P. serotina trees growing in various areas northeast of Mexico
City. Collected seeds were equally divided between the Michigan State University sour
cherry breeding program and the collaborating lab at the Universidad de Queretaro,
directed by Dr. Salvador Perez. At the end of this appendix are listed the P. serotina

contacts in Mexico.

66

Table 7. Collection information for Mexican P. serotina selections.

67

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825 2235
<2 830515 oom.~ >20 ES, :28; wok—E «882223 . 3.
823.5 8280
882 :omom canon
mm om ooo.~ >20 22>» m82< onzw 833 84 m.—
8235
825 22.2 :80 nomom
cm ow ooo.m >20 2S, .8 owum ontw 83mm 82 am
82.80
825 0:: 852 :28;
me on ooo.~ >20 22>) w82< chSw 83$ m3 :—
834 8280
<2 8582:: octm 88m 0:: 85.2 82m chCw 82882. . ma
mm om comm 28m .8: 85,2 on: 85,2 092% 88:83 Nu.
om 2 82m 28m 2.: 35m on: 35.2 882w 02282. :.
«you 8 Cb oW< 9.808 8 :5 «53m 58:88.00 8.5 82825 G—
52»: .883 gage—H 8282.30 853.30 £82..—

 

68

Table 7 (Continued). Collection information for Mexican P. serotina selections.

69

 

 

6080—2 8 20380 >8 2 Sta N000m 820223 .20 003002200 >2 200000230 0003 085023 a

 

 

 

 

 

 

 

 

 

 

 

 

 

2.2 m2 dmm 0. am mm >12. -08... 80,2 22 2.:
2<Emh<22
mam-Ego ad 92 2a m- mm M: 23.08:. 2832-80-2 .. 0w—
.00200mm 05 me
305— 0.806 005 802
82a :28 582 3 3 02 N mm mm 32 22 .5: ram 92
280303 8 2008222
2:28 B 5:0 no; mo 3 SM m 2 mm :2 22 .5: ram B
23:00 00,0208
9 088 0 00 20003 wd d._ mom m mm mm >22 22 .82. ram ND
8 M3 9;. m- on 8 257%: .3“. 22 . :-
mod ad omm m- 2.. en 08:.->22 .20“— 22 m.—
00 3 8m m- cm E 83.8: do» 22 8
0020229
02: 2000008 95 dd 0.2 dmm m- on em 08:.->22 80.2 2.2 E
ad 2.2 dmm m- R M: 23.08:. 2222-20-2 .. ma
o. 2 d. _ omm m- hm w _ >308... 2032-292 an.
083 >985
00—08 2 2000: 85 200G wd dad dmm m- nm 3 23-08:. 20822-802 =.
8895 he v.00: A800 2800 m>an 0.. 980,—. 0.. a80._.
130-— 858—08— 523 23:04 00.:— 30333 008.8% 025 025 A:
0082 8.80.50 2.— .u>< 2m .w>< «me-:2 85880—2 0uau0w>< 0003.”: 88:— >58:—

 

7O

P. SEROTINA (CAPULIN) CONTACTS IN MEXICO

Dr. Salvador Perez
Prol. Zaragoza 408
Jardines de la Hacienda

Queretaro, Qro. 76180
Mexico

Fax: 8-011-52-42-16-37-30

Juan Pablo

Mariano Monterde #44-A
Colonia Chapultepec Norte
Z.C. 58260

Morelia, Michoacan
Mexico

Tel: 8-011-52-43-14-59-81

Salvador Arteaga
RE: propagated capulins
via: Salvador Perez

Thomas Wallenmaier

US Dept. of Agriculture

Plant Protection & Quarantine
Rm. 228, International Terminal
Metropolitan Airport

Detroit, MI 48242

Tel: (313)924-7024

Jorge Rodriguez
email: Joroal@colpos.colpos.mx

71

APPENDIX B

ECUADORIAN P. SEROT INA GERMPLASM INFORMATION

72

The following appendix contains information regarding the identification of
Ecuadorian P. serotina Ehrh. Selections obtained from the Ecuadorian Germplasm
Institute. Ten Ecuadorian accessions were obtained as open-pollinated seed, presumably
collected from five trees, termed five collection ‘families’ (Ecu A 1-3, D 1-2, E 1, F 1-2,
and H 1-2) [c/o Dr. Raul Castillo,, Departamento Nacional de Recursos Fitogeneticos y
Biotecholigia, Casilla 17-01-340, Quito, Ecuador.] Uneven progeny from these five
Ecuadorian families resulted from poor seed germination. Identification of accessions
with the codes used at the Ecuadorian Germplasm Institute, as well as information

regarding seed germination are included.

73

Table 8. Ecuadorian P. serotina germplasm identification and germination information.

 

Accession Germplasm Institute No. Seeds

 

ID Identification Gerrninated
Ecu A DPRU 2188 3 / 5
EcuD DPRU2191 2/5
Ecu E DPRU 2192 1 /5
Ecu F DPRU 2193 2 / 5
Ecu H DPRU 2195 2 / 5

 

74

APPENDIX C

PRIMER PAIR SOURCES AND SEQUENCES

75

The following data table is a summary of information regarding the primer
sets used in the chloroplast fragment amplifications and the sequence tagged site
(STS) amplification of P. serotina selections for this thesis. Plant and laboratory
sources of each primer set, including sequence information are included for each

primer pair set.

76

Table 9. Primer pair sources and sequences.

 

 

 

<0 0<0 0<< <90 09 <0< <0< 8282 082 t. _< 0 M08
99 <<0 0<0 009 <09 909 9<0 8282 :82 3. 2 0 M08
0 090 <00 0<0 99< 09< 0<< 9<0 9<< 3282 08.2 a. _< m 820
0 90< 0<9 090 90< 099 0<0 9<< 090 8282 :82 3. _< a 820
0 <<< 99< 900 900 99< 090 900 <<0 8282 08.2 a. _< m 800
0 9<0 090 9<0 0<9 000 909 <90 00< 8282 8£< a. 2 a 800
0 <00 090 <0< 9<0 <09 090 <09 900 8282 :82 2. _< a 880
< 0<9 09< 090 0<< 099 9<0 <90 <00 8282 :32 ow ~< m 820
0 <00 090 <0< 9<0 <09 090 <09 900 8282 eo£< 8. _< m 88

0 900 9<< 090 0<9 0<< 099 <09 <00 8282 :82 mm 2 m 88 :08:
0 9<0 009 0<0 9<< 09< 9<0 8282 88 m _< x 808mm
9<0 090 <<0 <<0 9<< 000 8282 8:80 N9. 2 a 808mm
<09 00< 009 <0< 00< 00< 8282 88 R Z a 882mm
009 909 900 9<< 00< 000 8282 8:80 E _< 0 880mm

09 <<< 009 00< 00< 90< 009 8282 ESE 2 _< m 3 <0 8880

99 009 009 090 090 9<0 <<0 8282 82 cm 2 a «m <0 9mmam

< <9< 90< <90 9<9 <00 000 8 EON-E 8 _< m 83 8&2

< 099 <<0 990 00< <00 900 8 22 mm _< 0 SE 28:8 8

09 <9< 000 099 9<0 00< 909 8 8889 2 _< m m< 8&2

90 900 9<0 009 <<< 0<9 9<0 8 0:05 2 _< a m< 0389 8

am ............................................. am no HO DUHDOW OVCU DEMZ OOSOW Haw—hm

cocoa—com 30—032 Dai— Dm-H gosh-um ham HOST—m

 

78

APPENDIX D

RAW SEQUENCE TAGGED SITE (STS) DATA

79

The raw sequence tagged site (STS) data of four primer pairs producing 54
putative alleles for 66 P. serotina selections follows. The matrix is separated into

four primer pair selections.

80

Table 10. Data matrix for primer pair GA 34 containing 14 putative alleles numbered
according to their corresponding fragment lengths for each of the 66 P. serotina
selections assayed.

81

 

140 144 146 148 150 154 156 158 160 162 164 168 170 174

ID

0

BEAL

FU32
RG3
RG4
RG6
RG6
RG7
RGB
RG9

O
O
O

0

RG10
PH1
PH2
PH3
PH4
PH5
PH6
PH7
PH8
PH9

0

PH1O
P21
P22
P23
P24
P25
P26
P27
P28

P29

P210

82

Table 10 (Continued). Data matrix for primer pair GA 34 containing 14 putative alleles
numbered according to their corresponding fragment lengths for each of the 66
P. serotina selections assayed.

83

140 144 146 148 150 154 156 158 160 162 164 168 170 174

P32
P33
P34
P35
F36
P37
P38
P39

0
0
0

1

P310

0

ECUA1
ECUA2
ECUA3
ECUE1
ECUD1
ECUDZ
ECUF1
ECUF2
ECUH1
ECUHZ

0
0
0
0
0
0

0

0

0

84

Table 11. Data matrix for primer pair PSlZAOZ containing 12 putative alleles numbered
according to their corresponding fragment lengths for each of the 66 P. serotina
selections assayed.

85

0000100

0100000

0001000

0000000

0001111

0001100

0010000

0000000

0110100

1001010

0000001

1000001

150 154 156 158 160 162 164 168 170 172 176 178

 

000010000

000000000

000000000

000000000

000000000

000000000

000000000

101000000

111001000

010101111

000011000

00000000

00000000

00000000

00000000

00000000

00000000

00000000

11110100

00001011

00000001

00000001

0000

0000

86

Table 11 (Continued). Data matrix for primer pair PSIZAOZ containing 12 putative
alleles numbered according to their corresponding fragment lengths for each of
the 66 P. serotina selections assayed.

87

000000

0
O
O
O
O
O

O
0
O

000

O O O 0 0
O O O O O
O 0 O O O
O O O O O
0 0 O O 0
O O O O 0

150 154 156 158 160 162 164 168 170 172 176 178
1
O
1
O
O
1

000000

000000

000000

0

O

88

Table 12. Data matrix for primer pair 8681 containing 19 putative alleles numbered
according to their corresponding fragment lengths for each of the 66 P. serotina
selections assayed.

89

170 176 180 186 188 190 192 196 200 202 210 214 216 220 222 224 226 228 230

ID

1

1

PH2
PH3
PH4
PH5
PH6
PH7
PH8
PH9

O

PH10

Table 12 (Continued). Data matrix for primer pair B6B] containing 19 putative alleles
numbered according to their corresponding fragment lengths for each of the 66
P. serotina selections assayed.

91

170 176 180 186 188 190 192 196 200 202 210 214 216 220 222 224 226 228 230

ID

P21
P22
P23
P24
P25
P26
P27
P28
P29

P210

0
0
O
0
0
1
1
1
1
1
1

P32
P33
P34
P35
P36

Table 12 (Continued). Data matrix for primer pair B6Bl containing 19 putative alleles
numbered according to their corresponding fragment lengths for each of the 66
P. serotina selections assayed.

93

170 176 180 186 188 190 192 196 200 202 210 214 216 220 222 224 226 228 230

ID

P37
P38
P39

1
1
1
O
1
1
O
O
O
O
0

P310

ECUA1
ECUA2
ECUA3
ECUE1
ECUD1
ECUD2
ECUF1
ECUF2
ECUH1
ECUH2

Table 13. Data matrix for primer pair BlOH3 containing 9 putative alleles numbered
according to their corresponding fragment lengths for each of the 66 P. serotina
selections assayed.

95

130 132 138 140 142 144 146 150 152

ID

 

000010O0000000000000000000000000000010000
00000000000001010000000000010010100100110
00110101110110001100001101101101001000000
0000004|0000000000001000000000000000000000
00000001011110101110100010000000000100101
0000000010000101000000010011.1010110011.010
00000000000000000001011001000000000000000
00000000000000000000000000000000000000000

110111000000000OOOOOOOOOOOOOOOOOOO0000000

96

Table 13 (Continued). Data matrix for primer pair BlOH3 containing 9 putative alleles
numbered according to their corresponding fragment lengths for each of the 66
P. serotina selections assayed.

97

130 132 138 140 142 144 146 150 152

10000

00011

00100

00000

00011.

0
O
O
0
0

O

0000000000000000000

1010110100000000000

OOOOOOOOOOOOOOOOOOO

0000000001011001011

1100011110000110100

0011100000100000000

0000000000000000100

0000000000100000000

OOOOOOOOOOOOOOOOOOO
1M31121212
omumwwwwwww
3%M%%W%EMCCCCCCCCCC
PPPPPPPPPEEEEEEEEEE

98

"I1111111111111111“