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AF LP MAPPING AND QTL IDENTIFICATION FOR DAY
NEUTRALITY IN THE OCTOPLOID STRAWBERRY (Fragaria x
ananassa)

presented by

Cholani Kumari Weebadde

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AFLP MAPPING AND QTL IDENTIFICATION FOR DAY NEUTRALITY IN
THE OCTOPLOID STRAWBERRY (F ragaria x ananassa)

By

Cholani Kumari Weebadde

A DISSERTATION

Submitted to
Michigan State University
In partial fulﬁllment of the requirements
For the degree of

DOCTOR OF PHILOSOPHY

Genetics and Plant Breeding and Genetics Programs
Department of Horticulture

2005

ABSTRACT

AFLP MAPPING AND QTL IDENTIFICATION FOR DAY NEUTRALITY IN THE
OCTOPLOID STRAWBERRY (F ragaria x ananassa)

By
Cholani Kumari Weebadde

Day-neutrality in strawberry is a highly desirable trait to increase productivity. Day
neutral (DN) cultivars developed in California have not been successful in continental
regions of the US, because they are poorly adapted to high summer temperatures. Studies
on the inheritance of this trait are also contradictory and support a number of models
from single gene to quantitative inheritance. A linkage mapping approach was used to
determine if day neutrality is qualitatively or quantitatively inherited. Ampliﬁed
Fragment Length Polymorphic (AFLP) markers were used to build a genetic linkage map
from a segregating population created from a cross between the DN cultivar, ‘Tribute’
and the short day (SD) cultivar, ‘Honeoye’. One hundred and twenty seven progeny were
genotyped and 387 single dose restriction fragments (SDRFs) were mapped to obtain a
consensus map of 1310.7 cM with 42 linkage groups and an average marker density of
0.3 markers/CM. Individuals of the mapping population were observed for their ﬂowering
habit throughout the growing season in Michigan (MI), Minnesota (MN), Maryland
(MD), Oregon (OR) and California (CA), and quantitative trait loci (QTL) for day-
neutrality were identiﬁed from the phenotypic data collected at each of the locations. A
number of QTL were identiﬁed which were either shared or were location speciﬁc, but

none of these QTL explained over 40% of the phenotypic variation, indicating that

inheritance of day-neutrality is not regulated by a single, major gene. Of the QTL
identiﬁed for the eastern states (MI, MN, MD), one on linkage group 17 was found in all
eastern states, suggesting that a gene or genes regulating day neutrality is found in this
linkage group. However, only one signiﬁcant QTL was identiﬁed in the two western
states (OR and CA) and it was located on another linkage group (LG 7) in California.
These results suggest that different genes may be responsible for regulating day-
neutrality in eastern and western climates. The western climates of OR and CA, have
much milder temperatures than the eastern ones of MI, MN and MD during the
production season and generated higher percentages of DN progeny. This could be one
reason why DN cultivars developed in California do not perform as well in the eastern

climates.

I dedicate this work to three wonderful people in my life,
my parents and my husband,

for their undying support and encouragement towards my success.

iv

ACKNOWLEDEMENTS

I like to express my heartfelt gratitude to my major professor, Dr. Jim Hancock, for his
guidance, support and editorial assistance. I especially appreciate his sense of humor and

words of encouragement at times when they were most needed.

I appreciate the assistance from my committee members, Dr. Patrick Venta, Dr. Steven
van Nocker and Dr. Dechun Wang for their valuable suggestions on my dissertation and
helpful advice throughout the years. I would like to especially mention the contribution

made by Dr. Wang on my research and data analysis.

I would also like to thank past and present members of the Hancock lab, especially, the
lab manager Peter Callow and lab members Chris Owens, Sedat Serce, Chad Osborn and
Chrislyn Drake who made life in the lab a lot more enjoyable. Special thanks also go to
Tim Wentzloff for all his help on my project. Members of the Iezzoni lab, Bean lab,
McGrath lab, and Sink lab were also very helpful especially, Audrey Sebolt, Nat Hauck,

Jim Olmstead and Veronica Vallejo.

Special thanks also go to members of the ‘Biosafety team’ at MSU especially, Dr. Karim
Maredia. Dr. Maredia gave me the opportunity to work on International Agriculture,

trained me well to handle International projects and made my dream a reality.

I must also thank Dr. Barb Sears for all her help towards my mentored teaching project
and giving me the opportunity to tutor athletes at MSU which was a pleasant experience.
I also like to thank both Barb and Jim Hancock for giving me the opportunity to assist in

teaching their undergraduate Genetics class, which I enjoyed very much.

I would like to thank all my friends at MSU from Genetics, PBG, Microbiology, CMB
and IIA especially, Nat & Paula and Veronica for making my stay at MSU eventful and a
lot of fun. Special thanks also go to my two best friends, Uromi and Ravisha for being

there for me whenever needed.

I am also thankful to my loving parents who have taught me the importance of higher
education and cared for me always. My wonderful family and in-laws were always very
supportive. I would especially like to thank my wonderful husband Prabode, for being
there for me every step of the way encouraging me and lending a helping hand regardless
of whether it is in the ﬁeld, greenhouse or with my power point slides. This work would

not have been possible if not for his support.

vi

TABLE OF CONTENTS

LIST OF TABLES ........................................................................................................... viii
LIST OF FIGURES ........................................................................................................... ix
CHAPTER 1: LITERATURE REVIEW ............................................................................ 1
INTRODUCTION ............................................................................................................ 2
LITERATURE REVIEW ................................................................................................. 5
Physiology of ﬂowering in strawberry ........................................................................ 5
Genetics of ﬂowering in strawberry .......................................................................... 11
REFERENCES ................................................................................................................ 19
CHAPTER 2: BUILDING A GENETIC LINKAGE MAP FOR THE OCTOPLOID
STRAWBERRY USING AF LP MARKERS ........................................... 22
INTRODUCTION .......................................................................................................... 23
MATERIAL AND METHODS ...................................................................................... 27
Mapping population .................................................................................................. 27
DNA isolation ........................................................................................................... 28
Map construction and visual presentation ................................................................. 30
RESULTS AND DISCUSSION ..................................................................................... 31
REFERENCES ................................................................................................................ 42
CHAPTER 3: QTL IDENTIFICATION FOR DAY-NEUTRALITY IN THE
OCTOPLOID STRAWBERRY (Fragaria x ananassa) .......................... 45
INTRODUCTION .......................................................................................................... 46
MATERIAL AND METHODS ...................................................................................... 50
Obtaining phenotypic data ........................................................................................ 50
Genetic linkage map .................................................................................................. 54
QTL analysis ............................................................................................................. 54
RESULTS AND DISCUSSION ..................................................................................... 55
REFERENCES ................................................................................................................ 72

vii

LIST OF TABLES

Table 2.1: AF LP primer combinations and the number of polymorphic fragments
(PF) scored in a progeny population of ‘Tribute’ x ‘Honeoye’ segregating
for day-neutrality. The primers previously used by Lerceteau-thler
et. a1. (2003) are indicated with an asterisk... ................................................ 35

Table 2.2: Detailed presentation of the number of markers, length, marker density,
and gaps for each linkage group of the consensus genetic linkage map
constructed from the progeny population of the cross ‘Tribute’ x
‘Honeoye’ .............................................................................. 40

Table 3.1: QTL for day-neutrality that were detected in a segregating population
of ‘Tribute’ x ‘Honeoye’ grown in ﬁve different states. All QTL over a
LOD of 2.0 are listed, but an asterisk is used to denote QTL with a
LOD value significant at l % level. In Michigan, two separately
planted populations were evaluated, one that had been studied in
previous work (initial population - IP) and one newly reconstructed for
the present study (reconstructed population - RP). In Minnesota,
Maryland, Oregon and California, only RP was evaluated. The percent
phenotypic variation is indicated by the square value of the partial
correlation coefﬁcient (R2). QTL are named according to the
population, state, year and a number assigned in descending order
of the LOD value ...................................................................... 61

Table 3.2: Average minimum and maximum temperatures, and percent
day-neutral progeny observed at the various study locations used to
detect QTL for day-neutrality in a segregating population of ‘Tribute’
x ‘Honeoye’. The various study sites were located at Benton Harbor,
Michigan (MI), St. Paul Minnesota (MN), Beltsville, Maryland (MD),
Corvallis, Oregon (OR) and Watsonville, California (CA) ..................... 62

viii

Figure 2.1:

Figure 3.1:

LIST OF FIGURES

AFLP consensus genetic linkage map for the ‘Tribute’ x ‘Honeoye’

mapping population. Markers on the right are identiﬁed by the AF LP

primer combination, the fragment size and whether the marker was only
present in ‘Tribute’ (T), only present in ‘Honeoye’ (H) or, if marker

was present in both parents (B) ...................................................... 36

QTL for day-neutrality detected in the RP progeny of ‘Tribute’ x ‘Honeoye’
evaluated in Benton Harbor, Michigan, 2005. This population was newly
constructed for the present study (see text for details). Two QTL were
detected at the LOD threshold of 3.2 in linkage group 17 closest to markers
aggcatl 87 and atgcag205 ............................................................ 63

Figure 3.2: QTL for day-neutrality detected in the IP progeny of ‘Tribute’ x ‘Honeoye’

evaluated in Benton Harbor, Michigan, 2005. This population had been
studied in previous work (see text for details). One QTL was detected

at the LOD threshold of 3.2 in linkage group 8 closest to marker

atgctg249 ................................................................................ 64

Figure 3.3: QTL for day-neutrality detected in the RP progeny of ‘Tribute’ x ‘Honeoye’

evaluated in St. Paul, Minnesota, 2005. This population was newly
constructed for the present study (see text for details). Four QTL were
detected at the LOD threshold of 2.4 in linkage groups 7, 17, 20 and 28
closest to markers agtcag305, aggcatl 87, agacaal 74 and

agactc179 respectively ............................................................... 65

Figure 3.4: QTL for day-neutrality detected in the RP progeny of ‘Tribute’ x ‘Honeoye’

evaluated in Beltsville, Maryland, 2005. This population was newly
constructed for the present study (see text for details). Only one QTL was
detected at the LOD threshold of 4.01 in linkage group 17 closest

to marker aggcatl87... . 68

ix

Figure 3.5: QTL for day-neutrality detected in the RP progeny of ‘Tribute’ x ‘Honeoye’
evaluated in Corvallis, Oregon, 2005. This population was newly
constructed for the present study (see text for details). Two QTL were
identiﬁed with a LOD score above 2.0, but they were below the
5% LOD threshold of 3.13 ............................................................ 69

Figure 3.6: QTL for day-neutrality detected in the RP progeny of ‘Tribute’ x ‘Honeoye’
evaluated in Watsonville, California, 2005. This population was newly
constructed for the present study (see text for details). Only one QTL was
detected at the LOD threshold of 3.3 in linkage group 7 closest to
Marker agtcag225 ...................................................................... 71

CHAPTER 1

LITERATURE REVIEW

Introduction

Most cultivars of the strawberry, F ragaria x ananassa are classiﬁed as one of two types:
day neutral (DN) or short day (SD). Wild type SD plants (also known as June-bearers)
are facultative and initiate ﬂower buds either under short day conditions (less than 14hrs
of day length) or when temperatures drop below 15°C (Darrow, 1936). The DN
genotypes are photoperiod-insensitive and produce ﬂower buds approximately three
months after planting regardless of day length (Hancock, 1999). The DN genotypes also
produce ﬂoral meristems cyclically throughout the growing season, although the
production of ﬂoral meristems is inhibited at high temperatures. There are also long day
(LD) strawberry cultivars which ﬂower during the middle months of summer, but these

are rarely grown commercially and none have been released in the last 50 years.

F ragaria x ananassa is an autoallopolyploid with a chromosome number of 8x = 2n =
56. It was created by an accidental hybridization in the 17005 between F ragaria
virginiana and F ragaria chiloensis in a French botanical garden. There is little ﬁrm
evidence available as to how the octoploid species themselves arose, although most
investigators believe that the diploid strawberry species Fragaria vesca was a progenitor.
Photoperiod insensitivity is also observed in F. vesca, and is governed by a single
recessive gene at the SEASONAL FLOWERING LOC US (SFL) (Brown and Wareing,
1965; Cekic et.al., 2001; Albani et.al., 2004). As will be more fully described later,

photoperiod insensitivity in the octoploid appears to be controlled by more than one gene

and is at least partially under dominant control (Clark, 1937; Powers, 1954; Ourecky and

Slate, 1967; Ahmadi et.al., 1990, Sugimoto et.al., 2005).

About 25 percent of the world strawberry production is supplied by the United States
(US), of which nearly 80 percent comes from California, due in a large part to their
higher output per acre and their intense cultural systems (D. Bertelsen, 1995). Cultural
practices such as an annual planting system that use soil fumigation to eradicate pests and
the use of new and higher yielding DN varieties with an extended harvest season, have
contributed immensely toward Califomia’s dominance. Over 60 % of the Californian
strawberry production areas use DN cultivars (Hancock, 1999). Nursery plants are set out
each year in California in a winter (October-November) or summer planting system (late
J uly- September) and are replaced the following year. The plants are grown in clear
polythene mulch that warms the soil and stimulates early plant growth, giving California
a 12 month growing season, compared to about a 5 month growing season in other
strawberry growing states in the US (D. Bertelsen, 1995). In the Northwestern,
Midwestern and Eastern States of the US strawberries are grown as a perennial crop with

harvesting carried over 4 - 5 years after planting.

Day-neutrality was incorporated into modern commercial strawberry cultivars (F ragaria
x ananassa) by Bringhurst and Voth (1984), using a native genotype of Fragaria
virginiana ssp. glauca from the Wasatch Mountains of Utah. Compared to the traditional
short-day (SD) cultivars that ﬂower only once a year in spring, photoperiod-insensitive

DN cultivars ﬂower in any season three months after planting and cyclically thereaﬁer

throughout the growing season. When Californian DN genotypes are grown in
continental climates of northern America, their cyclic ﬂowering habit is hindered due to
summer heat. DN plants either remain vegetative failing to produce ﬂoral meristems at
30/26 0C day/night temperatures (Galletta et al., 1981; Dumer et al., 1984), or if they do
ﬂower during the middle months of summer, they have reduced yields and small, soft
fruits (Draper et al., 1981) with little economic value. Efforts have not been successful to
develop commercially acceptable DN cultivars for continental climates using the

Wasatch source of day-neutrality.

The low success rate at breeding for better DN cultivars for continental climatic regions
has stimulated increased interest in studying the inheritance of the trait and determining
the environmental cues associated with ﬂowering (Serce and Hancock, 2005a). A search
has also been initiated for new and stronger sources of day-neutrality for incorporation
into northern cultivars (Hancock et.al., 2001a). The goals of this study were to use a map
based approach to determine whether day-neutrality in octoploid strawberries is a
quantitative or a qualitative trait and to identify Quantitative Trait Loci (QTL) for day-

neutrality that can be used in marker assisted selection.

Literature Review

This literature review will focus on the various studies done on genetics and physiology

of ﬂowering in strawberry and how this information is related to the dissertation research.

Physician of flowering in strawber_ry

The most important factors that determine ﬂowering in the strawberry are temperature
and photoperiod. Darrow (1936) was the ﬁrst to measure the inter-relationship between
temperature and photoperiod in the production of runners and ﬂower buds of June
bearing strawberries. He discovered that short days stimulate ﬂower bud production at all
temperatures, although the most favorable temperatures for ﬂower production varied with
day length. He noted that a short day period of at least 14 hrs was necessary for inducing
ﬂower buds at 21 OC. He stated that photoperiods of much less than 14 hrs and
temperatures lower than 16 0C were most favorable for initiation of ﬂower buds, although
under these conditions ﬂower bud initiation proceeded at a slower rate. Darrow also
concluded that cultivars adapted to different geographical regions have their own
characteristic temperature day-length responses. Hartman (1947a, 1947b) veriﬁed
Darrow’s ﬁndings by discovering that ﬂower bud initiation in June bearing strawberries
was promoted if temperatures were less than 15 OC, even under long day conditions.
When temperatures were at 21 0C, he found that ﬂowers were only formed during short

days.

Downs (1955) compared the photoperiodic effect on ﬂowering in June bearing as well as
what he called everbearing types. He tested the effect of 11, 13, 15 and 17 hr day lengths
on ﬂowering. The June bearing types responded as expected with ﬂowers being promoted
under shorter day lengths (11 and 13 hr days) and being inhibited at longer day lengths
(15 and 17 hr days). The everbearers were able to produce ﬂower buds under all of the
above day lengths, but produced fewer ﬂower buds during short days. He did not
consider temperature regulation in his studies, as all his cultivars were subjected to the

same ﬂuctuating temperatures in a greenhouse.

Ito and Saito (1962) studied the effects of temperature and photoperiod on ﬂower
initiation in a June bearing strawberry under a number of controlled temperature and
photoperiod conditions. What was unique about their work was that they provided the
temperature and photoperiodic treatments in a phytotron with tight control of both
temperature and photoperiod, and aﬁer each treatment, transferred the plants to a
continuously illuminated greenhouse held at 24 0C. This avoided any effects that natural
temperature and photoperiod variation might have had on ﬂower formation. They found
that when the temperatures were as low as 9 0C, ﬂowers were formed regardless of day
length, while at temperatures of 17 0C or 24 0C, ﬂowers were only formed during short
days of 8 hrs. When they maintained plants under four different temperatures (9, 17, 24
and 30 0C) and seven different photoperiods (0, 4, 8, 12, 16, 20 and 24 hr day lengths),
they found that at 9 0C, 10 or more cycles of 8 - 24 hr day lengths stimulated ﬂower buds
whereas at 17 0C, ﬂower buds were only formed after 8 or more cycles of 4 - 12 hr day

lengths. Further increases in temperature to 24 0C increased the number of cycles of 4 -

12 hr day length necessary for ﬂowering to 10 and at temperatures of 30 0C, ﬂower buds
were not formed even with 20 cycles of short day conditions. They also found that
providing daily high temperatures of 24 0C for 12 hrs under continuous illumination
prevented ﬂower bud formation, even if the rest of the day was held at 9 0C. They
concluded that for optimal ﬂower bud formation in the strawberry, temperatures should
be below 12 - 14 0C and as the temperatures rise, shorter and shorter day lengths are

required to induce ﬂowering.

Durner et.al. (1984) tested temperature and night interruption (N 1) effects on ﬂower bud
formation in June bearing, DN and everbearing (LD) plants. LDs and NI inhibited
ﬂowering in the June bearers at 21 OC, and they concluded that ﬂowering in the June
bearers was regulated by photoperiod. On the other hand, ﬂowering in the everbearers
was enhanced by LD or NI, but was not completely inhibited by short days. The DN
cultivars ﬂowered regardless of photoperiod. It was also noted that the DN plants
behaved more like LD plants when the temperatures were at or above 22 oC/18 OC

day/night temperatures.

Nicoll and Galletta (1987) classiﬁed cultivars and selections by ﬂower response type to
see if ﬂowering response patterns had a relationship to morphological variation. They
studied plants belonging to ﬁve ﬂowering response types, which belonged to a continuum
of photoperiod and temperature response types: Strong DN plants (DNs), Weak DN
plants (DNw), Older everbearers (OEB), Reverted or very weak DN plants (DNr), and

June bearers (J B). As in the Dumer (1984) study, they used short day (9 hr) photoperiod

conditions along with a 3 hr NI to separate June bearers and everbearers. In their grth
chamber studies, the day/night temperatures were controlled at 22 0C/18 0C, whereas in
their greenhouse studies, temperatures were varied from a minimum of 19 0C to a
maximum of 35 0C. In both the growth chambers and the greenhouse, all the DNs, DNw
and the OEB ﬂowered at least once after NI, whereas ﬂowering in the DNr (only 27 %

ﬂowered) and J B genotypes (none ﬂowered) was limited by NI.

Based on their studies, Nicoll and Galletta classiﬁed cultivars into four photoperiodic
classes and three agricultural classes. The four photoperiodic classes were, strong DN
plants (including DNs), intermediate DN plants (including DNw and OEB), weak DN
plants (including the DNr) and the J B types. The three agricultural classes were single
cropping (including J B and DNr that do not produce any summer crop), intermediate
everbearer (including DNw and OEB that produce both summer and fall fruits in small
quantities) and strong everbearers (including DNs that produce summer and fall ﬁ'uits in
commercial quantities). They suggested that the terms DN and everbearer can be used

interchangeably if plants are classiﬁed in this manner.

Nicoll and Galletta also observed that the strength of everbearing was linked to the
growth habit of the plant and proposed three models to explain the different
photoperiodic types. For strong DN plants, they suggested that there is a high
inﬂorescence: crown ratio with few leaves intercalated between terminal inﬂorescences.
Every new shoot terminates growth by initiation of a ﬂower bud, resulting a smaller

meristem pool. In the intermediate DN plants, the branch crowns terminate in an

inﬂorescence. Therefore, the plant had many crowns and leaves with a 1:1 inﬂorescence:
crown ratio. For DNw, the main axis remains vegetative with axillary buds turning into
runners. These may produce a branch crown that rarely ends up in an inﬂorescence
during summer, although under short day conditions, the plants are able to produce

inﬂorescences on the main axis.

Manakasem and Goodwin (2001) also studied the responses of DN and June bearing
strawberries to temperature and day length. They used two day-length conditions (9 and
15 hrs) and three day/night temperature conditions (18/13, 21/16, and 30/26 0C) to study
the ﬂowering response to temperature and photoperiod, and used 15/10, 18/13, 21/16,
24/ 19, 27/22 and 30/25 0C day/night temperatures under natural daylight conditions to
test the effect of temperature on ﬂower initiation and development. As was observed in
the previous studies, a signiﬁcant cultivar x temperature x photoperiod interaction was
observed. They observed consistent ﬂowering in the DN plants over a wider range of
temperatures and photoperiods compared to the SD cultivars. As expected, the SD types
did not initiate ﬂowers during LDs, but ﬂowers initiated previously during SDs,
continued to develop well into the LDs. In the DN plants, ﬂower initiation occurred
during both LDs and SDs, resulting in continuous ﬂowering. A day/night temperature of
21/16 0C was found to be optimum for initiation of inﬂorescences in the SD cultivars,
although the greatest ﬂoral initiation occurred at 18/ 13 0C. In DN cultivars, they found
that the optimum day/night temperature for ﬂoral initiation depended on the cultivar and

varied with photoperiod.

Serce and Hancock (2005a) tested the effect of temperature and photoperiod on ﬂowering
and runnering of SD and DN representatives of the cultivated strawberry, F ragaria x
ananassa, and its wild progenitors, the scarlet strawberry, F ragaria virginiana, and the
beach strawberry, Fragaria chiloensis. When they measured the critical day length
(CDL) for ﬂoral induction of SD genotypes, a quantitative SD model ﬁt the cultivar
Honeoye, where the length of the photoperiod was signiﬁcantly correlated with ﬂower
number, but the other SD cultivars and wild genotypes did not show consistent trends.
The wild genotype Eagle 14 (F. virginiana), that had previously been categorized as a SD
type in the ﬁeld, produced the most ﬂowers during LDs in the growth chamber,
suggesting it is actually a LD type. They also found that there was a difference in the
number of ﬂowers produced by the different DN types in response to photoperiods of 8
vs.16 hr day lengths. Some DN plants produced the same number of ﬂowers regardless of
day length (eg. Frederick 9 of F. virginiana), while other genotypes produced the most
ﬂowers under LD conditions (eg. cultivar ‘Tribute’), and still others produced more
ﬂowers under SD conditions (eg. RH 30, also a scarlet strawberry). This indicates that
there is a continuous distribution in response to photoperiod among strawberry
genotypes. Serce and Hancock also tested the effects of temperature on ﬂowering of DN
and LD plants, in an attempt to select genotypes that can initiate ﬂowering under high
temperatures. All of the genotypes had signiﬁcantly lower productivity under high (30

0C) than low (18 0C) temperatures, except the old cultivar Fort Laramie.

In conclusion, the studies on the physiology of ﬂowering in the strawberry performed to

date, show a clear interaction between cultivar x temperature x photoperiod, and there is a

10

continuum in photoperiod response from short day to intermediate to fully DN plants.
Based on this range of response, it is likely that the photoperiod regulation of ﬂowering is

a complex genetic trait.

Genetics of flowering in strawberry

So far, no clear consensus exists on the inheritance of photoperiod insensitivity in
strawberry. Various attempts at studying the inheritance of this trait have resulted in
several different models: 1) regulation by a single gene (Ahmadi et. al., 1990 and
Sugimoto et.al., 2005); 2) regulation by two complementary genes (Clark, 1937); 3)
regulation by two complementary genes with interactions (Ourecky and Slate, 1967), and
4) regulation by at least 6 dominant and recessive genes (Powers, 1954). However, it is
difﬁcult to directly compare these studies, as they used different sources of day-
neutrality, dissimilar terminology to explain temperature/photoperiod interactions, grew
the plants under variant environmental conditions, and had diverse selection criteria for
DN plants. The older studies also considered all multiple cropping genotypes to be
“everbearing”, and they did not distinguish long-day ﬂowering genotypes from DN ones

that ﬂowered under both long and short days.

Clark (193 7) made the ﬁrst observations on the genetics of the everbearing trait in a New
Jersey breeding population. He observed different degrees of expression of the trait, with
some everbearers having continuous blooming patterns, while others produced only a few

ﬂowers during summer. He found that two of his parent plants (New Jersey No.1 and

11

New Jersey No.2) transmitted the trait to different percentages of progeny even when
they were crossed with the same individual. The phenotypic data he obtained by
combining several crosses was close to a 9:7 ratio, indicating that two complementary
genes may govern the everbearing trait. However, he believed that the breeding behavior
of the everbearing strawberries was not uniform and interactions with many genetic and

environmental factors could inﬂuence segregation patterns.

Powers (1954) studied the genetics of everbearingness in populations made by self and
cross-pollination of three selected everbearers (473, 475 and 476) and seven June bearers
(471, 472, 474, 477, 478, 479 and 4710). Altogether, he examined 10 self-pollinated and
45 cross-pollinated families. In the progeny populations of self pollinated 473, and the
cross between 473 x 475, he obtained a 3:1 ratio of everbearers to non-everbearers which
he concluded could have arisen if 473 and 475 were heterozygous for a dominant gene
conditioning everbearingness. In crosses between 473 and the June bearer types 471, 472,
474, 477, 478 and 4710, he obtained a 1:1 ratio, supporting the hypothesis that 473 was
heterozygous for a single dominant gene conferring everbearingness. However, he did
not observe the 3:1 ratio expected if the trait was regulated by a single dominant gene in
the cross between 473 and 476. When Powers combined all the progeny he obtained by
selﬁng 475 and 476 and by crossing 475 and 476, he observed a 37.5:62.5 ratio of June
bearers: everbearers suggesting 475 and 476 were heterozygous for two pairs of dominant
genes conditioning the trait rather than just one. Likewise, self pollinated progenies of
475 and 476, and the cross pollinated progeny of 475 and 476 did not produce the

expected 25 % non-everbearer types if both these parents were heterozygous for one pair

12

of genes conditioning the everbearing trait. When he crossed each of 475 and 476 with
the seven June bearers, he observed that the total progeny obtained ﬁt a 9:7 ratio of non-
everbearers: everbearers. He did not calculate if each of the above populations ﬁt the
same 9:7 ratio when taken individually; however, he suspected that in his combined
analyses numbers could have been balanced by individual crosses with too many or too
less everbearers because none of the crosses involving 475 or 476 were homogenous. He
further stated that some of the crosses involving these two parents deviated substantially
from simple genetic ratios and the only way the situation could be explained was to
assume that some of the non-everbearing types carry modifying genes. He also obtained
some everbearers from a number of self and cross-pollinations between J une-bearing

types, which would not be expected if inheritance was regulated by a dominant gene.

Powers concluded that the ﬂowering genes carried by the June bearers may have varied
in their dosage response, depending on the genetic background. He suggested that the
genes regulating SD in his June bearers may have been recessive to a dominant gene
carried by 473, but they may not have been recessive to those genes carried by 475 and
476. From these observations, he concluded that both dominant and recessive genes
conditioned the trait, and that there was an unequal contribution of dominant alleles
toward the trait. He further stated that the effects of all the genes are cumulative and that

there are at least 6 different pairs of genes with different dosage relationships.

Ourecky and Slate (1967) studied the segregation of the everbearing trait in progeny of

nine June bearing and four everbearing genotypes that were crossed in 25 different

13

combinations. They observed a wide range in percent everbearing progeny produced
either when the same everbearing type was crossed with a variety of June bearing types,
or when the same June bearer was crossed with different everbearing types. They also
found that only a few of the populations gave a 1:1 ratio of everbearing: June bearing
progeny, eliminating the possibility that a single gene governs the trait. When they
averaged the percent progeny obtained in the crosses involving each everbearing parent,
the percentage of everbearing progeny ranged from a 30.8 to 54.5 %. They hypothesized
that the trait was controlled by two dominant complementary genes with interactions.
They also suggested that there could be other genes for everbearingness not included in
their model, because a cross between one of their everbearing parents ‘Geneva’ and a

June bearer produced a higher percentage of everbearers than expected.

Ahmadi et.al. (1990) suggested in a study of a California breeding population that day-
neutrality in the octoploid strawberry is regulated by a dominant allele at a single locus.
He drew his conclusions from crosses made between DN and SD genotypes, selfs of the
hybrids and backcrosses to the short-day parents. He did not display the segregation
patterns of individual crosses and his chi square analyses were done on the combination
of all the crosses belonging to one type. For example, he tested whether the total of all
SD/SD x DN/SD and DN/SD x SD/SD crosses would result in an expected 1:1 ratio of
DN: SD progeny, all DN/SD x DN/SD crosses would result in an expected 3:1 ratio of
DN: SD progeny, all SD/SD x SD/SD crosses would not result in any DN progeny, and
all DN/DN crosses to either SD/SD or DN/SD would result in 100 % DN plants.

Although he used a large number of progeny and his ratios support single gene control,

14

there is a chance that by combining several populations belonging to one type, individual
crosses having too many DN plants got balanced by those that had fewer than expected,
as was suggested by Powers (1954). In a more recent study done on the California
breeding population using selfed progenies of 10 DN parents, Shaw (2003) found three
families that did not ﬁt an expected 3:1 ratio; two produced lower numbers than
expected and one produced higher number of DN plants than expected. This suggests that
day-neutrality is not controlled by a single dominant gene in the California breeding

population.

Ahmadi et. al. (1990) deﬁned day-neutrality in a number of different ways, depending on
the type of population they were evaluating: 1) plants that had ﬂowers on mothers as well
as runners during summer and fall under nursery conditions, 2) plants that ﬂowered three
to ﬁve months after germination, 3) plants that were continuously ﬂowering aﬁer being
established in the ﬁeld the fall of previous year and 4) classifying the parent plants from
observing their progeny obtained by hybridizing with the SD F. chiloensis and rating the

progeny as in # 2.

Single gene control for the everbearingness was recently found by Sugimoto et.al. (2005)
in Japan using the DN scoring method of Ahmadi et. al. (1990) and Barrit et. al. (1982)
where ﬂowers on mother and the ﬁrst runner plant were considered DN. When they
selfed populations of the everbearing genotype ‘Ever Berry’, and the June bearer
‘Toyonoka,’ they found that the progeny segregated in the 3:1 and 0:1 ratios of

everbearers: June bearers expected if ‘Ever Berry’ is heterozygous for the dominant gene

15

conferring the everbearingness. They also obtained the expected 1:1 ratio for the cross
between the ‘Ever Berry’ and ‘Toyonoka’ and identiﬁed Randomly Ampliﬁed

Polymorphic DNA (RAPD) markers linked to the everbearing gene.

Polygenic inheritance of day-neutrality was suggested by Hancock et. al. (2001b) to
explain the inheritance patterns observed in crosses of SD and DN representatives of

F ragaria virginiana from the wild with DN and SD Fragaria x ananassa cultivars. The
progeny of the crosses were planted at three different locations; Michigan, Minnesota and
Ontario and the plants were classiﬁed as DN if they ﬂowered under both SD and LD
conditions. A wide range in the percentage of DN progeny was observed among the
different SD F. x ananassa and SD F. virginiana families, which is not consistent with
control by a single dominant gene, particularly since most ratios deviated from a 1:1

ratio. In addition, they observed a signiﬁcant difference in the expression of day-
neutrality between the locations that indicated an environmental interaction. They found
that their segregation ratios ﬁt expected ratios of a single dominant gene control when the
DN F. x ananassa genotypes were crossed with SD genotypes, but when many of the DN
F. virginiana genotypes were crossed to the SD F. x ananassa genotypes the progeny
populations did not ﬁt monogenic ratios. This indicated that the two species were either
under different genetic control for the trait or that the trait is regulated in both species
quantitatively but as a threshold trait. To rigorously test whether multiple gerrnplasm
sources of day-neutrality exist, Hancock et al. (2001b) suggested testing segregating

populations of di-allele crosses among a broad range of DN and SD genotypes with

16

known responses to temperature and photoperiod. They also suggested the establishment

of a uniform method to characterize DN plants.

Serce and Hancock (2003) compared the 5 different methods commonly used to identify
DN plants (Ahmadi et. al., 1990; Barritt et. al., 1982) and found that the highest
proportion of DN plants was detected by growing plants in the ﬁeld under short and long
day conditions. In other work by Serce and Hancock (2005b), partial di-allele crosses
were made between 12 selected parental genotypes of the three octoploid strawberry
species to obtain 35 families of more than 50 individuals each that segregated for the day-
neutrality trait. Analysis of the ﬂowering habit revealed wide ranges in the percentage of
DN progeny in various families (30 — 87 % in DN x SD crosses and 22 — 93 % in DN x
DN crosses). These results suggested that day-neutrality is a quantitative trait and that

there is more than one source of day-neutrality in the native strawberry populations.

In conclusion, the conventional inheritance studies that have been performed to date
indicate that there is likely a small number of major genes regulating day-neutrality in
strawberries, but there are numerous other genes which have an inﬂuence on the trait
depending on the source of day-neutrality and the test environment. To clearly elucidate
the genetics of day-neutrality in strawberries, it will be necessary to study a diverse array
of the same segregating populations in several different environments (locations/year). It
would also be helpful to use a map based QTL approach to study the genetics of day-
neutrality, to identify how many genes are associated with day-neutrality and to calculate

their relative contributions to the phenotypic variability.

l7

The goal of this dissertation was to use such a map-based approach to identify the QTL
associated with one segregating population planted in several different environments. A
mapping population was selected from the previous Serce and Hancock (2005b) study to
build a genetic linkage map using Ampliﬁed Fragment Length Polymorphic (AF LP)
markers. This linkage map consisted of 387 markers and 42 linkage groups. The mapping
population was observed for ﬂowering during the SDs of spring and LDs of summer to
identify DN genotypes from that of SD types in Michigan in the year 2005. The same
mapping population was also observed in four other locations; Minnesota, Maryland,
Oregon and California, for their ﬂowering phenotype. The phenotypic data was analyzed
along with the molecular marker data to map Quantitative Trait Loci (QTL) for day-

neutrality.

The results indicated that different genes may be responsible for day-neutrality in the
eastern and western states, as a QTL observed in all the three eastern locations Michigan,
Minnesota and Maryland, was not seen in Oregon or California. Numerous other site-
speciﬁc QTL were identiﬁed. Since none of the QTL identiﬁed in any of the locations
explained over 40 % of the phenotypic variation, it appears that day-neutrality is not

controlled by a single dominant gene.

18

References

Ahmadi H., R.S. Bringhurst and V. Voth. 1990. Modes of inheritance of photoperiodism
in Fragaria. J. Amer. Soc. Hort. Sci. 115:146-152.

Albani M.C., Battey N.H., Wilkinson M.J. (2004). The development of ISSR-derived
SCAR markers around the SEASONAL FLOWERING LOCUS (SF L) in F ragaria
vesca. Theor Appl Genet 109:571-579.

Barrit B.H., R.S. Bringherst, and V. Voth 1982. Inheritance of early ﬂowering in relation
to breeding day-neutral strawberries. J. Amer. Soc. Hort. Sci. 107:733-736.

Bringherst RS. and V. Voth 1984. Breeding octoploid strawberries. Ipwa State J. Res.
58:371-381.

Brown T., Wareing RF. (1965). The genetical control of the everbearing habit and three
other characters in varieties of Fragaria vesca. Euphytica 14:97-112.

Berstelsen D. 1995. The US. Strawberry Industry. Commercial Agriculture Division,
Economic Research Service, US. Department of Agriculture. Statistical Bulletin
No. 914. http://www.nal.usda.gov/pgdic/Strawberry/ers/ers.htm

Cekic C., Battey N.H., Wilkinson M.J. (2001). The potential of ISSR-PCR primer pair
combinationsfor genetic linkage analysis using the SEASONAL FLOWERING
LOCUS in Fragaria vesca as a model. Theor and Appl Genet 103:540-546.

Clark J .H. 1937. Inheritance of so-called everbearing tendency in the strawberry. Proc.
Amer. Soc. Hort. Sci. 35:67-70.

Darrow GM. 1936. Interaction of temperature and photoperiodism in the production of
fruit buds and runners in the strawberries. Proc. Amer. Hort. Sci. 34:360-3 63.

Downs R]. and AA. Piringer, 1955. Differences in photoperiodic responses of
evberbearing and june-bearing strawberries. Proc. Amer. Soc. Hort. Sci. 66:234-
236.

Draper A.D., G.J. Galletta and H.J. Swartz. 1981. 'Tribute' and 'Tristar' everbearing
strawberries. HortScience 16:794-796.

19

Durner E.F., J .E. Barden, D.G. Himelrick and EB. Poling. 1984. Photoperiod and
temperature effects on ﬂower and runner development in day-neutral, junebearing
and everbearing strawberries. J. Amer. Soc. Hort. Sci. 109:396-40.

Galletta G.J., A.D. Draper and H.J. Swartz. 1981. New everbearing strawberries.
HortScience 16:726.

Hancock J .F. 1999. Strawberries. CAB International, Wallingford, UK.

Hancock J .F., P.W. Callow, A. Dale, J .J . Luby, C.E. Finn, S.C. Hokanson and K.E.
Hummer. 2001a. From the Andes to the Rockies: native strawberry collection and
utilization. HortScience 36:221-225.

Hancock J .F., J .J . Luby, A. Dale, P.W. Callow, S. Serce and A. El-Shiek. 2001b.
Utilizing wild Fragaria virginiana in strawberry cultivar development: Inheritance

of photoperiod sensitivity, fruit size, gender, female fertility and disease resistance.
Euphytica 126:177-184.

Hartman H.T., 1947a. Some effects of temperature and photoperiod on ﬂower formation
and runner production in the strawberry. Plant Physiol. 22:407-420.

Hartman H.T., 1947b. The inﬂuence of temperature on the photoperiodic response of
several strawberry varieties grown under controlled environment conditions. J.
Amer. Soc. Hort. Sci. 50:243-245.

Ito H. and T. Saito 1962. Studies on the ﬂower formation in strawberry plant. I. Effects of
temperature and photoperiod on the ﬂower formation. Tohoku J. Agr. Res. 13:191-
203.

Manakasem Y. and RB. Goodwin 2001. Response of day neutral and junebearing
strawberries to temperature and day length. J. Hort. Sci. & Biotech. 76 (5):629-635.

Nicoll M.F. and G.G. Galleta 1987. Variation in growth and ﬂowering habits of june
bearing and everbearing strawberries. J. Amer. Soc. Hort. Sci. 112:872-880.

Orecky D.K. and G.L. Slate 1967. Behavior of the everbearing characteristics in
strawberries. J. Amer. Soc. Hort. Sci. 91 :236-241.

Powers L., 1954. Inheritance of period of blooming in progenies of strawberries. Proc.
Amer. Soc. Hort. Sci. 64:293-298.

Shaw D.V., 2003. Heterogeneity of segregation ratios from selfed progenies demonstrate
polygenic inheritance for day neutrality in strawberry (Fragaria Xananassa Duch.).
J. Amer. Soc. Hort. Sci. 128:504-57.

20

Serce S. and J .F. Hancock, 2003. Assessment of day-neutral scoring methods in
strawberry families grown in greenhouse and ﬁeld environments. Turk. J. Agric.
For. 27:191-198.

Serce S. and J .F . Hancock, 2005a. The temperature and photoperiod regulation of
ﬂowering in Fragaria chiloensis, F. virginiana, and F. ananassa genotypes. Sci.
Hortic. 103:167-177.

Serce S. and J .F. Hancock, 2005b. Inheritance of day-neutrality in octoploid species of
Fragaria. J. Amer. Soc. Hort. Sci. 130:580-584.

Sugimoto T., Tamaki, K., Matsumoto, Y., Shiwaku, K., and K. Watanabe, 2005.

Detection of RAPD markers linked to the everbearing gene in Japanese cultivated
strawberry. Plant Breeding 124:498-501.

21

CHAPTER 2

BUILDING A GENETIC LINKAGE MAP FOR THE OCTOPLOID
STRAWBERRY USING AF LP MARKERS

22

Introduction

Most traits of agronomic importance are controlled by multiple loci and show a
continuous distribution in phenotype. Such traits are called complex or quantitative traits
(QTs) and efﬁcient breeding for them requires an understanding of the inheritance of the
genes involved in determining the trait. Often, such genes interact among themselves
and/or with the environment, which modiﬁes trait expression, complicating inheritance
studies. Furthermore, these genes can be scattered across the genome, making their

concentration into a single genotype difﬁcult.

Similar to all other breeders, strawberry breeders often struggle to improve QTs. One
such commercially important trait in strawberry that has proven recalcitrant is day-
neutrality or photoperiod insensitivity. The inheritance studies previously done for day-
neutrality have indicated that the trait may be under a wide array of control from single
gene to multi-gene (Clark, 1937; Powers, 1954; Ourecky and Slate, 1967; Ahmadi et. al.,
1990; Hancock et. al., 2001; Shaw 2003; Serce and Hancock, 2005 and Sugimoto et.al.,

2005)

In order to determine how many genes govern QTs, a linkage mapping approach can be
used in combination with phenotypic analyses. In this approach, a genetic linkage map is
generated using a mapping population that segregates for the QT of interest. This map is

then used to locate quantitative trait loci (QTL) that make a signiﬁcant contribution to the

23

expression of a complex trait, by identifying molecular markers on the map that co-
segregate with the trait of interest. Once chromosomal regions are identiﬁed that contain
the genes of interest, further ﬁne mapping can be done to ﬁnd markers that are closer to
the QTL. These can then be used in marker-assisted selection (MAS) in crop breeding

efforts.

Linkage mapping and QTL approaches are attractive to study the inheritance of day-
neutrality in strawberries for several reasons. In classical genetic studies, progeny
segregation ratios are used to determine how many genes govern a trait. Often due to
small sample size, numbers that depict contrasting phenotypes can ﬁt several different
genetic ratios complicating conclusions. Furthermore, QTs are often controlled not only
by a few major genes, but many minor genes that modify the phenotype depending on the
environmental conditions. When this happens, the ﬁnal phenotype that is scored can vary
greatly regardless of what the major genes are. In a QTL analysis, an association is
sought between the phenotype and the genotype depicted by molecular/DNA markers
(Collard et.al., 2005) and as a result, the number of genes determined to be associated
with the trait is not based solely on phenotypic ratios. It is even possible to detect genes
that have relatively limited affects on the phenotype, if the markers are closely linked to

the gene of interest.

The cultivated strawberry (F. x ananassa), is an octoploid species (2n = 8x = 56) and the
species ploidy level complicates linkage map construction, due to the possibility of

multisomic relationships and different loci carrying the same allele. However, the

24

limitations of genetic map construction in polyploids can be overcome by the use of
single dose restriction fragments (SDRF) (Wu et. al., 1992). An SDRF is a fragment
present in a single dose in a parent, and segregates as a single gene trait in the progeny. In
strawberry, when a fragment is present in a parent, it can be one of + ------- , ++ ------ , +++-
----, ++++----, +++++---, ++++++-- or +++++++- (where + and - signify presence and
absence of the marker respectively). Previous mapping experience with strawberry has
indicated that it has primarily disomic inheritance and about 120 individuals are adequate

to identify SDRFs (Lerceteau-thler et. al., 2003).

Complete linkage maps are only just beginning to emerge for F. x ananassa. Two of the
currently published maps were created with SDRFs of Ampliﬁed Fragment Length
Polymorphic (AF LP) markers and were used by Parisy (2001) and Lerceteau—Kbhler et.
al. (2003). Parisy (2001) used 518 SDRFs to generate female and male maps containing
44 and 46 linkage groups, respectively. The female map had a length of 1745 cM,
whereas the length of the male map was 1876.6 cM. Parents of his mapping populations
were not mentioned. The Lerceteau-thler group (2003) also developed separate female
and male maps of ‘Capitola’ x clone CF1116 using a total of 727 SDRFs, which
contained 43 linkage groups each, with lengths of 1604 cM (with 235 SDRFs) and 1496
cM (with 280 SDRFs), respectively. They were able to identify 30 and 28 linkage groups
on the female and male sides, and reported the genome sizes of the female to be 2870 cM
and the male, 1861 cM. Viruel et. al. (2002) crossed clones P1 and P5 (cultivar names
not mentioned) and used 177 Simple Sequence Repeat (SSR) and 123 Restriction

Fragment Length Polymorphic (RFLP) markers to develop a consensus map of 17

25

linkage groups and a length of 627 cM. He used 65 Prunus probes in his RF LP work, but
states that the probes generated low levels of polymorphism in strawberry compared to

Prunus. Additional information on where the probes and SSRs were derived was not

published.

If a QTL approach is going to be used to map the genes determining day-neutrality in
strawberry, we will need to examine a progeny population segregating for that trait. In the
currently published maps, where parents of the mapping population are indicated, there is
no mention that the population segregates for day-neutrality. Therefore, we cannot use
existing maps to do a QTL analysis of our trait of interest. The major goal of this research
was to develop a linkage map of a breeding population of F. x ananassa that was

segregating for day-neutrality.

We choose to build our map using AFLPs, as they are highly polymorphic, relatively
inexpensive to develop and only a few RF LP and SSR markers have been used in the
octoploid strawberry to date (Ashley et.al., 2003, Folta et.al., 2005). We included the
AFLP primer combinations used in the Lerceteau-thler et.al. (2003) study to search for
common markers. Our goal is to initially use AF LP markers to locate QTL for day-
neutrality, and then as more SSRs are developed, add these markers to build a dense map
that is also transferable across cultivars. AF LPs have also been used in other Rosaceous
crops such as apple to create saturated maps, with the inclusion of SSRs to make the
maps transferable among populations and species (Liebhard et. al., 2003). A few SSRs

have been reported in the diploid strawberry (Viruel et. al., 2002 and Sargent et.al., 2003,

26

Hadonou et. al., 2004), but their segregation patterns in the octoploid strawberry have not
yet been studied. We are currently collaborating with Dr. Kim Lewers at the fruit
Laboratory of the USDA in Beltsville, Maryland to develop strawberry SSRs from
published EST sequences. We also hope to determine if F. vesca and Prunus SSRs

segregate as SDRFs in our mapping population, to include them in our map.

Material and Methods

Mapping population

Six different populations were evaluated from the factorial experiment of Serce and
Hancock (2005) to identify the best population for mapping through selective genotyping.
All six populations were segregating for the day-neutrality trait. The crosses were,
‘Tribute’ x ‘Honeoye’, ‘Tribute’ x ‘Allstar’, ‘Tribute’ x Eagle 14 (native genotype of F
virginiana), Frederick 9 (native genotype of F. virginiana) x ‘Honeoye’, Frederick 9 x
‘Allstar’ and Frederick 9 x Eagle 14. Ten individuals, ﬁve day neutral (DN) and ﬁve
short day (SD) types, along with parents of each population were initially genotyped in

each population using 15 AF LP primer combinations.

The segregating mapping population that we selected to build the linkage map was
‘Tribute’ x ‘Honeoye’. This population had the highest number of polymorphic fragments

per primer combination (an average of 17) and the highest proportion of markers that

27

segregated in expected SDRF ratios (about 70 %). ‘Tribute’ is one of the two DN
cultivars released in the last 20 years for the eastern USA, while ‘Honeoye’ is the most

popular SD variety grown in the Midwestern and Northeastern USA.

DNA isolation

For reproducible AF LP analyses in strawberry, highly puriﬁed DNA is required as
strawberry DNA is often contaminated with large amounts of polysaccharides that tend to
inhibit digestion and ampliﬁcation reactions in the AF LP technique. Young leaves were
obtained from plants maintained in the greenhouse and were placed in coolers containing
ice before being transported to the lab. The Haymes (1996) method of DNA extraction
was used with the addition of two steps of chloroform / isoamyl alcohol (24:1)

extractions instead of one.

Leaf samples were lyophilized for 48 hrs after being frozen overnight at — 80 0C. 200 —
400 mg of lyophilized leaf tissue of each genotype was placed in a 2 ml tube with two 4
mm glass beads (Fischer scientiﬁc, Pittsburgh, PA) and 5 mg of polyvinylpyrrol-iodine
40 (PVP-40) (Sigma-Aldrich, St. Louis, MO). The samples were ground into a ﬁne
powder using the Fast prep FP20 (Savant Instruments Inc., Hallbrook, NewYork). The
powder was then transferred into a 15 ml centrifuge tube (Corning Incorporated, Corning,
NY) to which 6ml of pre-warmed extraction buffer (to about 65 0C) was added. After
mixing the contents well, the tubes were incubated in a water bath at 65 0C for an hour

with further mixing by inversion every 10 minutes. The samples were taken out of the

28

water bath and allowed to cool to room temperature before adding an equal volume of
chloroform / isoamyl alcohol (24:1). Then, the tubes were placed horizontally on a shaker
and were mixed for about 30 minutes. The aqueous and the organic layers were
separated by centrifuging at 6500 rpm for 20 minutes. The aqueous supernatant was
separated into a clean sterile 15 ml centrifuge tube (Corning Incorporated, Corning, NY)
and the chloroform/Isoamyl step was repeated. DNA was precipitated from the resulting
aqueous supernatant by the addition of an equal volume of ethanol-acetate solution (96
m1 of 200 % ethanol with 4 ml 3M sodium acetate solution) and gentle mixing by
inversion. The DNA precipitated as a white mass which was hooked using a sterile glass

rod, was washed twice with 500 pl of 70 % ethanol before being air-dried overnight.

The DNA pellet was dissolved in 150 pl of double distilled, autoclaved water and kept at
4 0C for two weeks before an RNase treatment (Roche- RNase DNase free) with 1 pl of a
10 mg/ml Rnase solution for each sample. DNA was quantiﬁed using the Hoefer DyNA
Quant 200 Fluorometer (Amersham Pharrnacia Biotech) and 100 ng/ pl dilutions were
made for each sample of DNA. The quality of the DNA was tested by running 5 pl of the
DNA sample with 10 pl water and 2 pl loading dye in a l % agarose gel for an hour

under 100 volts.

The Vos et al. (1995) protocol for AF LP analysis was performed on 127 individuals of
the segregating population using the modiﬁcations of Hazen et.al. (2002), Vallejo and
Kolkman (2002). 10 pl of each DNA sample (of 100 ng/ pl DNA concentration) was used

for each digestion reaction. 72 AF LP primer combinations were evaluated using EcoRI

29

and MseI (Table 2.1). One selective nucleotide from each of the primers was used in the
pre ampliﬁcation step and two additional selective nucleotides were used in the selective
ampliﬁcation step except for the combination, M + CG_ with E + AGT. After the
selective ampliﬁcation, 8 pl of the formamide loading dye (98 % forrnamide, 10 mM of
EDTA, 1 mg/ml xylene cyanol and 1 mg/ml bromophenol blue) was added to each PCR
product and all products were denatured for 5 minutes at 98 0C. After the denaturation,
the samples were immediately placed on ice to minimize renaturation before gel loading.
4.5 pl of each sample was loaded onto a 6 % denaturing polyacrylamide gel which was
pre equilibrated with pre heated l x TBE buffer. The gel was allowed to run for about
150 minutes after which the plates were separated and silver-stained. The plates were
then dried over night before the polymorphic fragments were scored over a light box. The
size of every polymorphic fragment scored was estimated by comparing it with 10 and 50

base pair ladders run on either side of the parents and progeny.

Map construction and visual presentation

Strawberry is highly heterozygous and is an outcrossing species and hence, a double
pseudo test cross strategy was used for mapping (Hemmat et. al., 1994). A total of 127
individuals of the cross ‘Tribute’ x ‘Honeoye’ was utilized in the segregation analyses.
The linkage mapping was done with those SDRFs that differed between parents and
segregated in a 1:1 (presence: absence) ratio and those that were present in both parents
and segregated in a 3:1 ratio. x2 analyses were performed to test goodness of ﬁt at 5 %

level and only markers that ﬁt were used in linkage analyses. Marker names were

30

selected to include the AF LP primer combination used, the size of the polymorphic
fragment and whether the fragment was present only in ‘Tribute’ (T), only in ‘Honeoye’

(H) or in both parents (B).

Joinmap 3.0 (Van Ooijen J .W. & R.E. Voorrips, 2001) was used to perform the linkage
analyses with a minimum LOD score of 3.0 and a maximum recombination fraction of
0.3. Map distances were calculated using the Kosambi map function and were expressed
in centi-Morgans (cM). Markers were excluded if their segregation pattern conﬂicted
with other markers in the same linkage group. MapChart software (Voopris, 1999-2002)

was used to draw the maps for the linkage groups.

Results and Discussion

Sixty nine AF LP primer combinations were used to genotype the 127 individuals in the
mapping population. Thirty-two of these primer combinations were among the 40
combinations used by Lerceteau-thler et. al. (2003). Table 2.1 presents a summary of
the primer combinations used and the number of polymorphic fragments (#PF) that were
generated from each primer combination. Of the 1065 polymorphic fragments scored,
279 markers were excluded from the map because they were resolved in fewer than 100
of the genotypes. Out of the remaining 786 markers, 539 or 69 % signiﬁcantly ﬁt the 1:1

or 3:1 ratios expected for SDRFs and were used to build the genetic linkage map. Of the

31

markers not included in the map, 16 % (247) signiﬁcantly ﬁt multiplex segregation ratios
[7:1 (49),11:3(12), 13:1 (18), 25:3 (29), 27:1 (1 3), 31:1 (5)], and 15 % did not ﬁt any
discrete segregation ratio. Thirty-eight markers signiﬁcantly ﬁt more than one complex

ratio.

Of the 539 SDRF markers, 383 segregated in a 1:1 fashion and 156 segregated in a 3:1
fashion. Three hundred and eighty seven of these markers were placed on the consensus
map, which consisted of 42 linkage groups. The rest of the SDRF markers were either not
linked to any of the linkage groups recognized or were not included because their
segregation pattern conﬂicted with other markers in the same linkage group at a LOD

score of 3.0.

While over 75 % of the AF LP primer combinations used by the Lerceteau-thler group
(2003) were included in our analysis, we were unable to locate any of their polymorphic
fragments in our population. It is possible that our parents did not carry the same
polymorphisms, but another reason might have to do with how we modiﬁed the Vos et.
a1. (1995) protocol. At the beginning of this study, several steps were taken to see if the
Vos et. al. (1995) protocol modiﬁed by Hazen et.al. (2002), Vallejo and Kolkman (2002)
would work well for strawberry. In this attempt, several different T aq polymerase
enzymes were tested (by Promega, Invitrogen etc.) to select the best enzyme for
ampliﬁcation reactions in the strawberry. Surprisingly, the banding patterns generated by

the different enzymes were not identical even when the rest of the protocol was

32

unchanged. Sigma T aq DNA polymerase was used by the Lerceteau-thler group and

we used Promega T aq DNA polymerase for our PCR ampliﬁcations.

Our map is more dense than the previous ones published for the octoploid strawberry.
Average marker density for our consensus linkage map was 0.3 markers/CM and the
length of the linkage map was 1310.7 cM. We also had an average of 9.2 markers per
linkage group (LG). Parisy (2001) developed female and male maps of 1745 and 1876.6
cM respectively and had one marker at each 7 cM with an average of 5.8 markers per
linkage group. Lerceteau-thler et.al. (2003) created maps of 1604 cM (female) and
1496 cM (male). They had an average of 5.5 markers per linkage group with an average
marker density of 8.4 ($7.9) markers/CM and 6.3 (2127.0) markers/CM for the two maps

(Kohler et al, 2003).

The longest linkage group of our map is LG 1, which is 122 cM in length with 31
markers (Figure 2.1 and Table 2.2). This group had a marker density of 0.25 markers/CM
and an average distance of 3.9 cM between markers. The shortest linkage group was LG

20, which had only two markers that mapped to the same locus.

The most dense of our linkage groups were LG 21, which had a marker density of 1.05

markers/CM, but only two markers.

33

Considering there are 28 linkage groups expected for the strawberry genome, our map is
far from being complete. It is likely that many of our linkage groups represent
fragmented portions of chromosomes and more markers are needed to link some of the
groups. We are currently developing SSR markers for this purpose in collaboration with
Dr. Kim Lewers in the Fruit Laboratory of the USDA in Beltsville, Maryland. However,
the objective of this work was to develop a sufﬁciently dense linkage map to determine
whether day-neutrality is a quantitative or qualitative trait. As will be described in
Chapter 3, this map was adequate to identify multiple QTL for day-neutrality in

strawberry.

34

Table 2.1: AF LP primer combinations and the number of polymorphic fragments (PF)
scored in a progeny population of ‘Tribute’ x ‘Honeoye’ segregating for day-neutrality.
The primers previously used by Lerceteau-Kohler et. al. (2003) are indicated with an

asterisk.

E = EcoRl primers, M = Msel primers

 

Primer E+aa- #PF E+at- #PF E+ac- #PF E-I-ag- #PF

 

M+ca~ aag/cag 11 ata/cac 19 acclcag 13 agtlcaa 19
atg/cag‘ 17 acclcac 11 agtlcat' 8

atg/cac“ 15 acclcag 13 agtlcag“ 14

atg/caa“ 15 acalcaa 16 agalcat‘ 11

acalcag“ 12 agg/cag 12

acclcaa‘ 19 agc/cag 14

acalcat‘ 8 agglcat‘ 17

act/cag 14 agalcag“ 4

aga/caa" 17

agalcac‘ 17

agg/caa 23

M+ct- aag/cta 16 atg/ctg“ 18 acc/cta 19 agalctc 16
aag/ctt 22 atg/cta“ 19 acclctg‘ 8 agtlcta‘ 25
aag/ctc 27 ata/ctc‘ 27 act/eta 17 agalctt* 22
aac/ctg 17 atg/ctt‘ 20 acc/ctt 14 agtlctc“ 8
aag/ctg 8 ata/ctt 21 act/ctt 18 agg/ctt 10
atg/ctc" 17 act/ctc 15 agg/ctg 15

acc/ctc* 8 agalctg‘ 24

act/ctg" 19 agc/ctt 1 1

acalctc“ 1 3 agtlctg‘ 1 3

 

 

agg/ctc 1 1
agg/cta 4
agalcta‘ 18
agc/ctg‘ 8
agt/ccc 10
age/ctg“ 10
M+cc- ata/ccg 18 act/cog 10
ata/cca 20
ata/cct 18
ata/ccc 27
M+cg- ata/cgc 7 agt/cga 8
atg/cg-* 19 agg/cga 14
ate/cit 7

 

35

Figure 2.1: AF LP consensus genetic linkage map for the ‘Tribute’ x ‘Honeoye’ mapping
population. Markers on the right are identiﬁed by the AFLP primer combination, the
fragment size and whether the marker was only present in ‘Tribute’ (T), only present in

‘Honeoye’ (H) or, if marker was present in both parents (B).

 

 

LG2
0.0 agtcta332T LG3
12.3 agtct a400T
13. 1 a cot 362B
14.9 agtctagZWST 0.0 8900112498
LG] 15. 1 ag acaa298 B 0.7 actcth40H
17, 3 aagcnzsz'r 0.8 agacaa265H
18.9 actcta32 7T :13: 2199 £13121 359%
atgcta173T 28.8 zggoocga-gZBTQH 6.0 agtocc157B
20.4 atactc176B 9.5 econ-91247
23. 3 agtcta172T 11.2 aggcga283H
25.4 agacac231B 11.9 accczg124H
atgctc1 86T 26. 3 agoctt1 2213 26. 0 3390:9353ng
agtcga14OT 28.9 agacat1868 31-2 agac 9
a gtcta133T 3% aggiggg:
. ag
2:22:31;ng 32. 4 ata cac362H
aagcag2o7T 33 9 atgwc44OH
. a agcag49 8H 34. 1 ata oct94T LG4
‘ aggcagzsoT 34.3 aggcat1048
.1 actctc247B 34.6 atgcao442H
atgcac1848 35. 6 atgctc2628 agacat315H 0 0 actct157 0B
‘ atgcac1858 35 71 3130032648 34 899698212 H
atacgc243T 36. 9 j Lagocag183H 6.1 atactc242H
atacoc128B 38-4 Lat9010159T 6 9 aggcag435T
atgcac153T 39.0 j Lagtm a403 H 15.6 agacaa347 H
atgcac284T 39. 4 L atgog-Z 83H 16. 3 atgmc204T
atactt470 B 39. 91 L atactt451 T 22. 3 actctcSZSHH
atgcg—293B 40. 9 ‘ Latgcth40H 25.1 ataoca187
atgcg-2288 41.1 4 Late occ121T 25. 8 accottsgglgs
atgctt140 H 42.41 LatgcacAZZB 26. 2 acacag
atgctg108T 42. 7 . L atgcao4188 33. 2 atgog.217:6HB
atgcg-224B 45. 2 ‘ Lag acaa450 H 35. 9 accmc1 10H
a“mam 49. 8 4 aa gcta350T 42.6 as ocag 7
58.54 Lacccags40H 45.1 atauc45 H
”0th 7‘” 46. 5 accua113H
a‘a‘ﬁmz H 48. 2 aggctt278B
agtcta128B 52.0 ataogc315H
agcctt141T 54.9 aagcta224H

 

36

Figure 2.1 (Cont’d).

LG5

0.0— — ata ctc365T
6.0— —atgog-118T

O
11.1 —r_ agmgzzsr

27.4 — — actctt117T

37.9—~— agtwg305T

 

 

52.0—5— agtwa230T

LG9-l

ag actt199T
atgctgt4OT
atg c1951 5T
actcta38 OT
atg <2 c2 56T
atg ca c2 57T
acacaa1 54T
accctc21 4T
acccta21 1T
aagctaSOO B
atgog -2 34T

LG12

0. O ag actc330H

0. 1 atgctc21 2H

8. 9 agacat182T
14.5 agacaa137 H
15.0 ag acth73 B
16.9 ag acta231 B
17. 7 accca 3298B

aagctc2 30H
accdc136H

§NF°9°>l9792QQNO
OOwNVCOJAO'JU’IAO

N4

LG6
0.0 actcta3888
18.5 atactc2593
28.8 atgd9535H
34.9 ag gcag271 H
38.0 aagcagtBQB
LG9-2
0. 0 ag gcga329H
3. 7 agacaa163 B
5 2 actcta154H
15.2 atactc295H
LG13
O. 0 atgcacSSST
3. 8 acccaa262T
6.2 actctg226T
9. 1 actcthZ 5T
12.0 aggcga122T
16.0 ata ctt193T
20. 8 atactc193T

 

 

 

 

 

 

0. 0 ag acac2 59H
0.0 agtctc193H 0- 7 319°91'ng
1 .9 agtca g323T
7, 0 ag actc2 52H 2, 5 actctcS45T
16.7 ataeacsaoH 5'5 39 303334“
7.1 actcagZ 638
18.9 agtcta449H
8. 4 accctai 398
23.7 ag acaa230H
10. 2 atact622 28
25.2 atgdg198H t 282T
28. 7 agactc3538 1 1-3 a 909
31.0 atgdc179H 17-2 393011114“
19. 2 accdc22 4T
4o_5 atgcta119H 20.7 atact022 5H
22.9 atg cth49T
26.3 ata oca2 53T
36.6 ag actt163T
LG10
0. 0 ataw$40H
1.3 atactc3468 LGll
3. 2 ataoca24ZB
3. 9 agacaa212 B
4. 6 ag acta285 H 0. 0 0— ag acthZST
6.5 agtwa110H 3_3 ——agactc1 38H
7. 9 ataocc928
10. 8 atacat$30T
15.1 acccaa130H 15.4 “3“"335T
15.5 aagctt162T 19.5 agtﬁa373T
19.4 atg (1945011 24. 9 ataxa126T
24.9 agtcta141T 28.0——-agtcaa363T
34.2 ——— agtdc145T
41.3— —atgog-112T
47. 8 acacaa1 348
51.3“ ~atgctt138T
LG14-1
0. 0 agalcta-1
15.2 act/cagZ LG14-2
16.3 ata/cog7
19.9 acclcag-6 aoc/cagG

37

0.0 accdg 220H
5.7 actcta2288
7.8 ataccgz438

Figure 2.1 (Cont’d).

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

    
 

L
0.0 agoctg338T
0.
0.0 agactc380H 0-0 899039268T 0 “@3310” 6 6 3300,91,,”
W \ .5.
. \_/-a . agac
10:6 “was” 14.7\_/ aagcta370T :33 2:36:32? 18.2\._/ acccta244T
.M—\. ' [1"1‘
18.8 “”3155“ 16.0/__ atactc1658 21‘0 ata (104048 20.3 actca98243T
19.2 \atacac164T
24.1 N,_./ actctg129H “455*.
25.9 #1:» ataogc190H 27-7 39‘” 30.3 agtcaa440T
30.3 agtoga143T
LG25
LG19 LG20 LG21
0.0 accwcgggi
10.6 agacta
0.0 ataoct440T 0.0—e—lacaca1309Hac10392563 $3 232333;: 16.2 ‘ ataocg163T
4.6 agtcta208T ' 30.4 I agaca0238T
36.7 aggca1245T
13.1 atgctt383T 52.1 ‘ I agactt187H
52.3 it agtcaa355T
18.7 actctg167T 54.7 , .1 atgctc272T
55.9 \ / atgogZBOH
57.6 aggcga505T
LG23 LG24 58.9 I ataoca168H
60.7 accct1233B
61.9 1 acccag124H
0‘0 ta t555 62.4 , 1 actc10390H
LG22 3'2 22:313ng 3 cc 8 63.9 I aggcga295T
' agtcag 3608 66.2 I 35303322563?
66.8 5 aaog
67.7 w: atgcag1528
0'0 39“”“28” 68.0 g agtctc2058
LG27 69.4 E aagcta203B
70.3 I 8193601848
00 ataoca101B 72.5 I atactc172T
' 73.0 aa gcthOS B
2‘5 zalcm9223“ 2.6 8996891913 LG28 75.3 ! actcag159T
2226:1231: «941931
- 7. at caa21 T
7.4\ /aggcat187T 8 2 g
14.7\ /aaoctgz66H 0.0 aagctaZ30H
14.8 ‘1/ atan0368T
17.3\ /ataczc378B
18.1 \=/ atgctt148T
LG26 20.9 atgcth48T
31.1 7:§ ataott415T 25- 5 " 81391259“
0.0 accma193H 31.6 / \aggctt1908
3.7 agt-t193H 34.9 /:\atgc29205T 37.2 aggcga102H
9.3\—/agacac370H 40.2 6430002133 48.5 agoctg103H
12.5 acacat318B 42.0 3180081743 51.4\ /aaoctgz76H
16.0"‘~acacaa151H 42.3 3680892303 54.4§:/atguc209H
46.0 aagct9119T 55. 3 ata ocg3 30H
23-7 39289375“ 48.4 attics-118T 55.9 acacaa296H
50.6 atactt167T 56.8 atgca0208H
54.4 aagcta2958 57,2 accma1458
55. 9 ag 3033333 B 63.2 ag acaa1 15 H

38

Figure 2.1 (Cont’d).

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

L629
0. 0 ag actt238H
21. 5 ata ocg1 82H
35.3 aagcto4 20H
39. 1 agtca a209 H
44. 7 atactc31 8H
LG33
0. 0 ~ actctg187H
3. 3 aa gct1290H
22,8 \__/- agactg181 H
23.8 /—\ agactg1158
36.9 acacagZ 97H
LG37
0. 0 atgca c1 87H
9. 9 acccaa212 H
16.6 actcag362H
35. 1 atg c2 a400 H

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

LG30 LG31 L332
0. 0 aa gct1297T
. 1 4. 4 atg mc350T
0.0 agtcga 3608 2.8 :6: 29:33 (1)ng 5_ 1 atg @034“
4.8 ace-£92688 8.1 atgctt253T
12.2 agactg186T
LG35
LG34 LG36
0. 0 aa gcttZ94H
0. 0 ag acta253 B 0. 0 atacao405T
6' 9 ag “3391 65” 4. 5 ataoch 51T
13;):(22333218433: 14.9 atactt245H 11.6 EEC ataocc201T
21.3 actcth64H ”'6 39mm”
24. 7 \_./ actcta197T 28- 0 3130091 72”
I — \
27'1 “”923” 35.7 aagcta425H
33. 5 atg cth35T
40. 5 atg 01921 3T 45.0 v atgctc198H
LG40
LG38 LG39
0.0 acccta 141 H
. T
0. O acacaa1 348 0 0 agtocc229 8.8 agg cttZ38H
5. 7 aa gcta266T 6. 3 ata oct40 0T
10. 4 acorn a395T 10- 4 3130102593
18.0 aa gctg73T
LG42
LG4 l 0. 0 agacttZBST
2'3 2:331? 11.0 agtctttSQT

39

Table 2.2: Detailed presentation of the number of markers, length, marker density, and

gaps for each linkage group of the consensus genetic linkage map constructed from the

progeny population of the cross ‘Tribute’ x ‘Honeoye’.

 

Linkage group

 

 

 

 

1 2 3 4 5 6 7 8 9-1 9-1 10
AFLP markers 31 38 11 19 6 5 9 15 11 4 12
Length in cM 122 58.5 31.2 54.9 52 38 40.5 36.6 24.6 15.2 24.9
Marker density 0.25 0.65 0.35 0.35 0.12 0.13 0.22 0.41 0.45 0.26 0.48
MarkersIcM
Average distance 3.9 1.5 2.8 2.9 8.7 7.6 4.5 2.4 2.2 3.8 2.1
between markers
Largest gap 35.3 12.3 14.1 8.7 16.3 18.5 9.7 10.3 12.6 10 5.5
Linkage group 11 12 13 14-1 14-2 15 16 17 18 19 20
AFLP markers 10 9 7 5 3 6 7 6 6 4 2
Length in cM 51.3 26.4 20.8 19.9 7.8 25.9 30.3 27.7 30.8 18.7 0
Marker density 0.19 0.34 0.34 0.25 0.38 0.23 0.23 0.22 0.19 0.21 0
Markersch
Average distance 5.13 2.93 2.97 3.98 2.6 4.32 4.33 4.62 5.13 4.68 0
between markers
La_rgest m 12.1 8.8 4.8 15.2 5.7 8.2 11.1 7.9 10.5 8.5 0

 

40

Table 2.2 (Cont’d).

 

 

 

 

 

Llnkage group 21 22 23 24 25 26 27 28 29 30 31
AFLP markers 2 2 2 2 26 6 23 13 5 2 2
Length in cM 1.9 21.5 3.5 7.6 87.2 23.7 56.9 63.2 44.7 4.8 2.0
Marker density 1.05 0.09 0.57 0.26 0.3 0.25 0.4 0.21 0.11 0.42 1
MarkersIcM
Average distance 0.95 10.75 1.75 3.8 3.35 3.95 2.47 4.86 8.94 2.4 1
between markers
Largest gap 1.9 21.5 3.5 7.6 15.4 7.7 7.3 20.5 21.5 4.8 2
Linkage group 32 33 34 35 36 37 38 39 40 41 42
AFLP markers 5 5 7 7 4 4 3 4 2 2 2
Length In an 12.2 36.9 40.5 45 13.6 35.1 10.4 18 8.8 4.4 11
Marker density 0.4 0.14 0.17 0.16 0.29 0.11 0.29 0.22 0.23 0.45 0.18
MarkersIcM
Average
distance 2.44 7.38 5.79 6.42 3.4 8.78 3.47 4.5 4.4 2.2 5.5
between
markers
Largest gap 4.4 19.5 14.1 9.3 7.1 18.5 5.7 7.6 8.8 4.4 11

 

41

References

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in Fragaria. J. Amer. Soc. Hort. Sci. 115:146-152.

Ashley M.V., Wilk J .A., Styan S.M.N, Craft K.J., Jones K.L., Feldheim K.A., Lewers
K.S., Ashman TL. 2003. High variability and disomic segregation of
microsatellites in the octoploid Fragaria virginiana Mill. (Rosaceae). Theor Appl
Genet URL http://www.springerlink.corn/app/home/content/asp?wasp=hpb9j dh. ..

Basten C.J., Weir BS. and Z.B. Zeng 2002. QTL Cartographer, Version 1.16.
Department of Statistics, North Carolina State University, Raleigh, NC.

Clark J .H. 193 7. Inheritance of so-called everbearing tendency in the strawberry. Proc.
Amer. Soc. Hort. Sci. 35:67-70.

Collard B.C.Y., Jahufer M.Z.Z., Brouwer J .B., and B.C.K. Pang 2005. An introduction to
markers, quantitative trait loci (QTL) maooing and marker-assisted selection for
crop improvement: The basic concepts. Euphytica 142:169-196.

Folta K.M., Staton M., Stewart P.J., Jung S., Bies D.H., Jesdurai C. and D. Main 2005.
Expressed sequence tags (ESTs) and simple sequence repeat (SSR) markers from
octoploid strawberry (F ragaria x ananassa). BMC Plant Biol. 5:12.

Hadonou A.M., Sargent D.J., Wilson E, James C.M., and D.W. Simpson 2004.
Development of microsatellite markers in F ragaria, their use in genetic diversity
analysis, and their potential for genetic linkage mapping. Genome 47:429-438.

Hancock J .F., Luby J .J . , Dale A., Callow P.W. , Serge S. and A. El-Shiek. 2001.
Utilizing wild F ragaria virginiana in strawberry cultivar development: Inheritance
of photoperiod sensitivity, fruit size, gender, female fertility and disease resistance.
Euphytica 126:177-184.

Haymes KM. 1996 mini-prep method suitable for a plant breeding program. Plant
molecular biology reports, 14-280-284.

Hazen S.P., Leroy P. and R. Ward 2002. AFLP in Triticum aestivum L.: patterns of
genetic diversity and genome distribution. Euphytica 125 (1): 89-102.

Hemmat M., Weeden N.F., Manganaris AG. and D.M. Lawson, 1994. Molecular
markers linkage map for apple. J. Hered. 85:4-11.

Lerceteau-Ktihler E., Gue'rin G., Laigret F. and B. Denoyes-Rothan. 2003.
Characterization of mixed disomic and polysomic inheritance in the octoploid

42

strawberry (Fragaria Xananassa) using AF LP mapping. Theor. Appl. Genet.
17:619-628.

Liebhard R., Koller B., Gianfranceschi L. and C. Gessler 2003. Creating a saturated
reference map for the apple (Malus x domestica Borkh.) genome. Theor. Appl.
Genet. 106: 1497-1 508.

Orecky D.K. and G.L. Slate 1967. Behavior of the everbearing characteristics in
strawberries. J. Amer. Soc. Hort. Sci. 91:236-241.

Parisy V. 2001. Establishment of the strawberry, F ragaria x ananassa Duchesne, genetic
map; search for molecular markers of agronomic characters. The French Institute
for Agranomic Research (INRA), Fruit and wine research station, Bordeaux,
France. (Abstract) URL http://genetics.biology.kyushu-
u.ac.jp/pgen/cv/vipar/fragaria%20proj ect.html

Powers L., 1954. Inheritance of period of blooming in progenies of strawberries. Proc.
Amer. Soc. Hort. Sci. 64:293-298.

Sargent D.J., Davis T.M., Tobutt K.R., Wilkinson M.J., Battey NH. and D.W. Simpson
2004. A genetic linkage map of microsatellite, gene-speciﬁc and morphological
markers in diploid F ragaria. (Abstract) Theor Appl Genet (in press) URL
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=disply&db=pubmed&uid=1 52
90052&dopt=abstract

Serce S. and J .F. Hancock, 2005. Inheritance of day-neutrality in octoploid species of
Fragaria. J. Amer. Soc. Hort. Sci. 130:580-584.

Sugimoto T., Tamaki, K., Matsumoto, Y., Shiwaku, K. and K. Watanabe, 2005.
Detection of RAPD markers linked to the everbearing gene in Japanese cultivated
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Vallejo V. and J. Kolkman (2002),
http://www.css.msu.edu/bean/PDF/AFLP_protocol.pdf

Van Ooijen J .W. & R.E. Voorrips, 2001. Join map Version 3.0, Soft ware for the
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Viruel M.A., Sénchez D. and P. Arus 2002. An SSR and RFLP linkage map for the
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Conference, San Diego, CA. (Abstract)

Voorrips R. E. 2002: MapChart, Software for the graphical presentation
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43

of linkage maps and QTLs. J. Heredity 93, 77—78.

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J ., Peleman J ., Kuiper M. and M. Zabeau, 1995. AFLP: a new technique for DNA
ﬁngerprinting. Nucleic Acid Research 23:4407-4414.

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fragments. Theor Appl Genet 83:294-300.

44

CHAPTER 3

QTL IDENTIFICATION FOR DAY-NEUTRALITY IN THE
OCTOPLOID STRAWBERRY (Fragaria x ananassa)

45

Introduction

Day-neutrality was ﬁrst introduced into to short day (SD) strawberry cultivars in
California by Bringhurst and Voth (1984) using a native genotype from the Wasatch
Mountains of Utah. Over 60 % of the California production areas are now planted with
day neutral (DN) cultivars to extend the production season. Wild type SD plants or J une-
bearers initiate ﬂower buds either under short day conditions (less than 14 hrs of day
length) or at lower temperatures of below 15 0C (Darrow, 1936). The DN plants are
photoperiod insensitive and ﬂower early (ﬂoral meristems produced after a shorter

vegetative phase) and cyclically throughout the growing season.

The cyclic ﬂowering habit of DN genotypes would be useful to extend the harvest season
in other regions of the US. However, when the DNs developed in California are grown in
continental climates of North America, they remain primarily vegetative when day/night
temperatures are above 30/26 0C and any fruit that are produced during the warmest

months of summer are small and soft (Galletta et.al., 1981, Dumer et.al., 1984).

Attempts at studying the inheritance of day-neutrality in octoploid strawberries have
resulted in several different models being presented including: 1) regulation by a single
dominant gene (Bringhurst and Voth, 1978, Ahmadi et. al., 1990, Sugimoto et. al., 2005);
2) regulation by dominant complementary genes (Clark, 1937 and Orecky and Slate,

1967); and 3) quantitative inheritance (Powers, 1954; Hancock, 2001c; Serce and

46

Hancock, 2005). However, these studies are difﬁcult to compare as they utilized different
sources of day-neutrality, dissimilar terminology to explain the different
temperature/photoperiod interactions, a variety of environmental conditions and a wide

array of selection criteria for what constituted DN plants.

Studies done by Hancock et. al. (2001) investigated the inheritance of day-neutrality in
strawberry by crossing a diverse group of SD and DN representatives of F ragaria
virginiana from the wild with SD and DN F ragaria x ananassa cultivars. Cultivar
representatives were used from all the US breeding programs and the progeny were
evaluated in three different locations; Michigan, Minnesota and Ontario. In their crosses
of DN F. virginiana x SD F. x ananassa, most of the progeny ratios deviated
signiﬁcantly from a 1:1 ratio, indicating that photoperiodic insensitivity is not controlled
by a single dominant gene in these populations. They detected a wide variation in the
percentage of DN plants produced by each DN F virginiana parent and DN progeny
were generated from crosses of SD F. virginiana and SD F. x ananassa; all these
observations are consistent with polysomic inheritance. A signiﬁcant difference in the
expression of day-neutrality was also observed across locations, indicating a strong

environmental component.

In a comparison of the ﬁve techniques commonly used to identify DN genotypes, Serce
and Hancock (2003) found that the highest percentage of DN plants was discovered when
segregating populations were grown in the ﬁeld for a full season, and plants were

classiﬁed as DN, only if they ﬂowered under both short and long day conditions. Serce

47

and Hancock (2005) also made partial diallele crosses between 12 selected SD and DN
genotypes of California cultivars, Eastern cultivars and a group of wild genotypes that
had previously been shown to produce different frequencies of DN progeny. The
segregating populations were analyzed for whether they ﬂowered under SDs (<14 hr) in
the spring (before 30th of May) and under LDs (>15 hr) in the summer (after 24‘'1 July).
They found a wide, almost continuous range in the percentage of DN progeny produced
across families, from 30 — 87 % in DN x SD crosses and from 22 - 93 % in DN x DN

CI'OSSCS.

Serce and Hancock (2005) made a number of additional observations that suggested that
day-neutrality in octoploid strawberry is inherited as a complex trait. Overall, their DN x
DN crosses produced 71 % DN progeny, signiﬁcantly lower than the expected 75 % if
day-neutrality was controlled by a single dominant gene. Their SD x DN crosses
produced 58 % DN progeny, which is signiﬁcantly higher than the 50 % expected. The
selfed DN progeny from the strong DN cultivar ‘Tribute’ produced the highest
percentage of DN progeny (88 %) compared to any other DN x DN cross. In addition,
different percentages of DN plants were produced when different DN parents were
crossed to the same SD genotype, and some of the DN sources appeared to be stronger in
their ability to generate DN progeny. They also found that none of the DN x DN crosses
produced 100 % DN progeny as would be expected if they were homozygous for a
dominant gene, and only half the families ﬁt a segregation ratio of a single dominant gene

model.

48

In another study of the genetics of day-neutrality using California breeding populations,
Shaw (2003) selfed populations of 10 DN F. x ananassa genotypes and observed a
continuous range in the percent DN progeny produced by the various selfed DN parents.
He also found that 3 of the families did not segregate in the 3:1 ratio expected for
heterozygous DN parents carrying a single dominant gene that controls the trait. Out of
the three families, two produced lower numbers of DN plants than expected, while the
third produced an excess. He concluded that day-neutrality in the California breeding

population is not controlled by a single locus.

Based on these most recent ﬁeld studies of a wide range of germplasm, we hypothesized
that day-neutrality is governed by a few major genes (controlling a major component of
the phenotype) and many minor genes or modiﬁers that alter the phenotype depending on
the environment, as has been detected for many quantitative traits (Patterson et. al.,
1991). To directly test this hypothesis, a linkage mapping approach was used to
determine if there are multiple QTL for day-neutrality that map at different locations. A
suitable segregating population for day-neutrality for this approach was selected from the
previous partial diallele crossing experiment by Serge and Hancock (2005), and was used
to develop a genetic linkage map. Phenotypic data on ﬂowering was obtained on a
weekly basis in Michigan during both SD and LD conditions and were analyzed with

marker data for detecting QTL for day-neutrality/cyclic ﬂowering trait.

It was realized that the QTL associated with day-neutrality would need to be veriﬁed

before using them in strawberry breeding efforts. There are several ways to verify QTL:

49

l) by testing the same mapping population in multiple environments, 2) searching for the
same QTL in independent populations, 3) developing near isogenic lines (N ILs) that
differ only in the QTL region, 4) through high-resolution QTL mapping and 5) by
cloning and transforming the putative QTL into varieties lacking them to see if the
introduced QTL gives expected expression. Since strawberry is a highly heterozygous,
outcrossing species, we are unable to develop near isogenic lines, and our map was not
sufﬁciently dense to perform high-resolution QTL mapping or cloning. Therefore, we
veriﬁed the QTL using multiple environments and independent populations. In this paper,
we report on the identiﬁcation of QTL for day-neutrality in Michigan, and our efforts to

verify the QTL in Minnesota, Maryland, Oregon and California.

Material and Methods

Obtaining phenotypic data

The ﬁrst 62 individuals of the mapping population (initial population or IP) were
obtained from a previous study (Serce and Hancock, 2005). Mother plants were dug from
the ﬁeld in the fall of 2003, potted in 15 cm diameter pots, and maintained in an unheated
greenhouse over the winter. During summer of 2004, we expected to collect runner plants
from these mother plants to be set in the ﬁeld in fall of 2004 at Benton Harbor, MI.
However, most of the mother plants did not produce any runners, so we propagated the IP

using a single crown separated from each of the mother plants. The plants were then

50

grown in a heated greenhouse over the winter and set in the ﬁeld at Benton Harbor in

spring of 2005.

Because the IP did not have sufﬁcient individuals for map construction (Chapter 2), we
re-constructed the mapping population (re-constructed population or RP) using the same
parents in spring and summer of 2003. Anthers of fresh ﬂowers of the pollen parent,
‘Honeoye’ were collected into 2.5 cm diameter petri dishes and were allowed to dehisce
overnight at room temperature in the lab. Some of the petri-dishes containing the pollen
were sealed with paraﬁlm and stored at —16 0C for future use. In the female plant
‘Tribute’, mature ﬂower buds were selected for crossing that were about to open and all
stamens were removed using sharp tweezers to avoid self pollination. Crosses were made

using a camel hairbrush that was used to transfer the pollen on to the pistils of the ﬂower.

Seeds were hand picked from harvested fruits and were placed on trays containing sterile
moist soil. The trays were then kept in a growth chamber maintained at 4 0C with
continuous ﬂuorescent light for a period of 3 months, by which time the seeds were
beginning to germinate. The trays were transferred to a growth room with a temperature
of about 18 0C with continuous ﬂuorescent light to enhance germination of seeds. Once
the seedlings were at four to six leaf stages, plants were potted in 7.6 x 7.6 cm cell packs
and allowed to grow in the growth room until they were acclimated for potting in a green
house. A total of 360 plants were recovered from the seeds. The plants were transferred
into 14 x 12 x 12 cm pots in April 2004 and were maintained in the greenhouse, where

they were allowed to runner.

51

About 10 runners were collected from each of the mother plants during the ﬁrst week of
July 2004. Runners of each of the genotypes were separated from each other and planted
into 2.5 x 2.5cm cell packs ﬁlled with a commercial soil mix and were maintained for
two weeks in a mist house to induce roots. The rooted runner plants were then transferred
to a greenhouse for acclimation and were held for two weeks with watering every day. In
the last week of July, each of the runner plants with 2 - 4 leaves, were packed into
separate plastic Ziploc bags with a tag identifying the plant number. The plants were held
at 12 0C over night and were shipped to four different strawberry growing states in the
US; Minnesota (St.Paul), Maryland (Beltsville), Oregon (Corvallis) and California
(Watsonville), for testing in those sites the next day. The plants were sent to our
collaborators of this project; James Luby (R342, Alderman Hall, Department of
Horticultural Science, University of Minnesota, 1970, Folwell Ave., St. Paul, MN
55108), Kimberly Lewers (USDA-ARS, Fruit Lab, R 210, 10300, Baltimore Ave.,
Building 010A BARC- West, Beltsville, MD 20705-2350), Chad Finn (USDA-ARS,
Horticultural Crops Research Laboratory, 3420, NW Orchard Ave., Corvallis, OR 97330)
and Tom Sjulin and Jill Bushakra (Driscoll Strawberry Associates inc., 404, San Juan

Road, Watsonville California 95077).

In Michigan, one set of runner plants were potted in 14 x 12 x 12 cm pots with a
commercial soil mix and were held in the greenhouse at East Lansing for three weeks
before they were planted in a Completely Randomized Design at the SWMREC Research

Farm of Michigan State University at Benton Harbor. They were set at 1.2 m x 1.2 m in

52

the ﬁeld and were planted in the fall of 2004. The IP was planted at the same site with the

same dimensions in the spring of 2005.

The RP were planted in a similar ﬁeld design in Maryland and Oregon in the fall of 2004
after being potted and held in a greenhouse for 3 weeks. In Minnesota, plants were also
set in the ﬁeld after being maintained in the greenhouse for 3 weeks, but at 0.45 m x 0.45
m spacing on black plastic. In California, the plants were maintained in a screen house
for 4 months before they were set in the ﬁeld at 0.36 m x 0.36 m in a green semi-opaque

plastic mulch.

Flowering response was evaluated by recording the presence of open ﬂowers at weekly
intervals at all locations starting the ﬁrst week of May 2005 and continuing until the end
of August 2005. Plants were considered to be DN, if they ﬂowered both under SD and
LD conditions in the ﬁeld as suggested by Serge and Hancock (2003). Since ﬂowers
initiated under SD conditions may still develop well into the LDs (Manakasem and
Goodwin, 2001), we considered a plant to be day-neutral only if it ﬂowered after the 15th
of June, allowing over a month for any SD initiated ﬂowers to develop. At all locations,
days become longer than 14 hrs after the ﬁrst week of May. From previous studies, it is
known that strawberry plants take from 7 - 22 days to initiate ﬂowers depending on the
temperature (Hartman, 1947b). The photoperiod requirement of each genotype at each
site was rated by assigning either number 1 or 2, depending on whether they ﬂowered

only during SDs (1), or ﬂowered under both SDs and LDs (2).

53

Genetic linkage map

The genetic linkage map previously generated using the 62 progeny of the IP and 65 of
the RP was used to detect QTL (Chapter 2). This map consisted of 387 markers and 42

linkage groups.

QTL Analysis

The phenotypic data and molecular marker data for each individual of the mapping
population was analyzed using the WIN QTL CARTOGRAPHER software (Basten, C.J.,
B.S. Weir and Z.-B. Zeng, 2002). Since this software is only capable of analyzing one
type of maker data at one time, three different maps were constructed, two maps each
using 1:1 segregating markers that were present in ‘Tribute’ and absent in ‘Honeoye’ and
vice versa, as well as a map using 3:1 segregating markers that were present in both the
parents. For each of these three maps, QTL mapping was performed as composite
interval mapping (CIM) available with the WIN QTL CARTOGRAPHER (Basten, C.J.,
B.S. Weir and Z.-B. Zeng, 2002) software program. CIM gives better precision for QTL
compared to the interval mapping (IM) method since CIM tests for QTL in one interval at
a time and so, has the ability to control background effects of other QTL out side of the
interval being tested. For all of the analyses, model 6 (standard model) of CIM was used
with a window size of 10 cM. The background control marker number was kept to 5
markers, which were detected through a forward and backward stepwise regression. The

LOD threshold for detecting QTL for each of the locations was tested by performing

54

1000 permutations. Two data sets were analyzed in Michigan; (1) phenotypic data on
only the 65 progeny of the RP planted in the fall of 2004 and (2) phenotypic data on only

the 62 IP planted in the spring of 2005.

The phenotypic variation explained by each QTL was obtained from the output of the
analysis from the WIN QTL CARTOGRAPHER software (Basten C.J., B.S. Weir and
Z.-B. Zeng, 2002). The estimation for the proportion of the phenotypic variation
explained by each QTL was made using the square value of the partial correlation

coefﬁcient (R2).

Results and Discussion

In the previous studies done by Serge and Hancock (2003 and 2005), genotypes were
considered DN if they ﬂowered during the SDs of spring and the LDs of summer from
July 24th to August 24th. However, there was a chance that some DN genotypes were
misclassiﬁed that ﬂowered before this cutoff date. Some DN plants that are more
sensitive to higher temperatures may begin ﬂowering for a second time before the end of
July when the longer day-lengths begin, but temperatures are not as high as in late July.
Therefore, in 2005, we took data on ﬂowering on a weekly basis from the ﬁrst week of

May until the last week of August. While less than 3 individual genotypes were found in

55

Michigan and Minnesota that ﬂowered before July 24th, this number ranged from a 5 - 15

at the other locations.

Table 3.1 gives a summary of the QTL detected in Michigan, Minnesota, Maryland,
Oregon and California. Any QTL that was observed above a LOD score of 2.0 is
reported; however, an asterisk is used to denote those QTL that were above the threshold
LOD score determined by the permutation test. The LOD thresholds determined at the 1
% signiﬁcance level for Michigan, Minnesota, Maryland, Oregon and California were
3.2, 2.4, 4.01, 3.8 and 3.3 respectively. All the QTL associated with day-neutrality were

derived from the cultivar ‘Tribute’.

In the eastern states (Michigan, Minnesota and Maryland), a number of QTL were
identiﬁed that were associated with day-neutrality, and all of these states shared a QTL
on linkage group 17. For RP in Michigan, two QTL were identiﬁed (Table 3.1, Figure
3.1), both of which were on linkage group 17 and had R2 values of 26.1 and 21.9 %.
These two QTL were closest to markers aggcat187 and atgcag205. One QTL was
detected for IP in Michigan over a LOD threshold of 3.2 (Table 3.1, Figure 3.2). This
QTL was located on the linkage group 8 closest to marker atgctg249 and was responsible
for 25 % of the total phenotypic variation. In Minnesota, 4 QTL were detected above the
LOD threshold of 2.4 that were located in the linkage groups 7, 17, 20 and 28 (Table 3.1,
Figure 3.3). The QTL on linkage group 7 was closest to marker agtcag305 and had an R2
value of 14.43 %. The QTL detected on linkage group 17 was closest to the aggcat187

marker and was responsible for 20.1 % of the phenotypic variation. R2 values of 11.48

56

and 13.04 % were obtained for the QTL detected on the linkage groups 20 (closest to
marker agacaa174) and 28 (closest to marker agactcl 79). One QTL was detected in
Maryland on linkage group 17, which was closest to marker aggcat187 (Table 3.1, Figure

3.4) and was responsible for 36 % of the phenotypic variation for day-neutrality.

These data suggest that there is likely a gene or genes on linkage group 17 that regulates
day-neutrality in eastern US climates. It is unlikely that it is a single major gene, as it was
responsible for at most, 36 % of the phenotypic variation. Several other location speciﬁc
QTL were also identiﬁed, indicating the regulation of day-neutrality is multigenic, with a
strong environmental interaction. A QTL on linkage group 17 was not identiﬁed in the
Michigan IP, but it was handled quite differently to RP, being planted in the spring rather

than the fall and as crowns instead of runners.

Only one signiﬁcant QTL was identiﬁed in the segregating population evaluated in the
western states (Oregon and California) of the US. A highly signiﬁcant QTL was
identiﬁed in linkage group 7 in California (Table 3.1, Figure 3.6) that was closest to
marker agtcag225 and was responsible for 22 % of the phenotypic variation. A QTL in
this linkage group was also identiﬁed in Minnesota and Michigan RP. No signiﬁcant
QTL were detected in Oregon at the threshold LOD score of 3.8 (Table 3.1, Figure 3.5),
although one on Chromosome 14 (closest to marker aggctg217) was just below the 3.13

LOD threshold cut off point at the 5 % signiﬁcance with a LOD score of 3.1 1.

57

It is not known why fewer QTL were identiﬁed in the western than eastern states of USA,
but it can be speculated that the different temperature patterns found in these regions may
have played a role. F rom studies done on the physiology of ﬂowering, it is known that
there is a strong temperature/photoperiod interaction that determines ﬂowering in the
strawberry. When average high temperatures are below 15 0C, all genotypes tend to
behave in a photoperiod insensitive manner (Hancock, 1999). Michigan, Minnesota and
Maryland had average mid-summer temperatures ranging above this threshold from 16 to
18 OC and they had similar percentages of DN progeny (49.23 %, 50 % and 48 %
respectively). Oregon and California had much lower temperatures during the LDs of
mid-summer that were below the threshold (11 and 13 OC) and they had a much higher
percentage of DN plants (80 and 87 %). Twenty-seven of the genotypes that did not
ﬂower under LDs in the eastern states ﬂowered in the west because of this difference in

temperature patterns.

This suggests that different genes may be responsible for regulating day-neutrality in the
eastern vs. western states of the US. There may be a gene or genes regulating
temperature insensitivity that must be present to allow for DN ﬂowering in the warmer
eastern climates. The QTL identiﬁed on LG 17 may be such a gene. In the cooler western
states, this gene may not play a role in the expression of day-neutrality, as temperatures
are too mild to need its expression for the trait. This may contribute to the reason why the
California DN cultivars have not performed as well in eastern strawberry growing states
than western ones. Depending on environmental conditions, different loci in the genome

of strawberry may become important for ﬂowering.

58

In conclusion, day-neutrality does not appear to be under single major gene control, as we
did not observe any one QTL that explained more than 40 % of the phenotypic variation
at any location. Day-neutrality may indeed be quantitative in nature; as we identiﬁed
numerous QTL with modest effects ranging from 15 — 40 %. It is possible that a strong,
dominant locus regulating day-neutrality does exist, and we didn’t uncover a marker
tightly linked to it as in Sugimoto et. al. (2005); however, the regulation of day-neutrality
still has to be considered under polygenic control, as multiple QTL were detected at the

various locations.

Considering there are 28 chromosomes in the strawberry genome and we have identiﬁed
a higher number of linkage groups (42), many more segregating markers will need to be
added to the map to have complete genome coverage. Better map coverage with more
AFLP markers and SSR markers, will allow us to more precisely estimate QTL locations
and may result in the identiﬁcation of more genes regulating day-neutrality. An
evaluation of our total population of 422 individuals will also lead to better map
resolution. We have already started genotyping the remaining of the 422 individuals of
the mapping population with AFLPs and the entire RP has been phenotyped in the ﬁeld.
The population will again be observed for their weekly ﬂowering habit next year (2006)
in Michigan, allowing us to test the reproducibility of the QTL in multiple years. In
addition, work has been initiated by one of our collaborators, Kim Lewers, to genotype

the population using SSRs developed from strawberry EST libraries. These data will

59

allow us to produce a much denser map and transfer our QTL results between different

segregating populations.

60

Table 3.1: QTL for day-neutrality that were detected in a segregating population of
‘Tribute’ x ‘Honeoye’ grown in ﬁve different states. All QTL over a LOD of 2.0 are
listed, but an asterisk is used to denote QTL with a LOD value signiﬁcant at 1 % level. In
Michigan, two separately planted populations were evaluated, one that had been studied
in previous work (initial population - IP) and one newly reconstructed for the present
study (reconstructed population - RP). In Minnesota, Maryland, Oregon and California,
only RP was evaluated. The percent phenotypic variation is indicated by the square value
of the partial correlation coefﬁcient (R2). QTL are named according to the population,

state, year and a number assigned in descending order of the LOD value.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Closest
Environment QTL LG Marker LOD R2
Michigan LIP) lPMlOS-l * 8 atgctg249 3.3644“ 25%
Michigan (RP) RPMIOS-l * l7 aggcatl87 6.252" 26.10%
RPM105-2“ l7 atgcag205 5.3382“ 21.90%
RPM105-3 17 atﬂg248 2.6339 12%
RPM105-4 aMZZS 2.3618 19.70%
RPM105-5 atacca253 2.3497 1 1.50%
RPM105-6 agtctal 72 2.1343 7.90%
Minnesota RPMN05-1* 17 aggcat187 4.8158* 20.10%
RPMN05-2" 7 aM305 2.7796“ 14.43%
RPMN05-3* 28 agactc l 79 2.7679* 13.04%
RPMN05-4“ 20 agacaa174 2.7569“ 1 1.48%
Maryland RPMDOS-l * 17 aggcat l 87 6.4563"l 36%
RPMDOS-Z 29 atgcagl 35 2.4505 1 1.70%
RPMD05-3 1 acacag380 2.3314 13%
Oregon RPOROS-l 14 aggtg217 3.1 127 15%
RPOR05-2 l7 atggggZOS 2.8089 14.60%
California RPCAOS- I " 7 agtggs 4.6717" 22%
RPCA05-2 10 agactg228 2.6086 1 1%
RPCA05-3 5 atacgc286 2.2766 8.60%

 

61

Table 3.2: Average minimum and maximum temperatures, and percent day-neutral

progeny observed at the various study locations used to detect QTL for day-neutrality in a

segregating population of ‘Tribute’ x ‘Honeoye’. The various study sites were located at

Benton Harbor, Michigan (MI), St. Paul, Minnesota (MN), Beltsville, Maryland (MD),

Corvallis, Oregon (OR) and Watsonville, California (CA).

 

 

 

 

Location MI MN MD OR CA
% DN plants 49.23% 50% 48% 80% 87.30%
Temp. SDs (April)
average min 5°C 5°C 4°C 4°C 7°C
average max 17°C 16°C 17°C 16°C 19°C
Temp. LDs (July)
average min 16°C 18°C 17°C 11°C 13°C
average max 29°C 29°C 28°C 27°C 22°C

 

62

Figure 3.1: QTL for day-neutrality detected in the RP progeny of ‘Tribute’ x ‘Honeoye’
evaluated in Benton Harbor, Michigan, 2005. This population was newly constructed for
the present study (see text for details). Two QTL were detected at the LOD threshold of

3.2 in linkage group 17 closest to markers aggcat187 and atgcag205.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

lmLG17
- N 0.: ts u: m u on up 8
0.0 agg/cat-12 n - n: . r r r . .
!s /

o

S"
8.1 ata/cac7
11.5 atg/ctt12
14.4 atg/ctg10

+ MI05

24.5 ata/cttB

E
28.7 atg/cag10 8

1'6
41.9 ata/c1119
47.9 aag/ctg-7

 

 

 

63

Figure 3.2: QTL for day-neutrality detected in the IP progeny of ‘Tribute’ x ‘Honeoye’
evaluated in Benton Harbor, Michigan, 2005. This population had been studied in
previous work (see text for details). One QTL was detected at the LOD threshold of 3.2

in linkage group 8 closest to marker atgctg249.

lmLGB

 

 

0.0\ /atgct9249 o T '1’ 3’ A

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

0.7 atgog-92

1.9 _ agtm9323

2.4 / \ actct0345

5. 5 agacaa340
1 1.4 atgog-2 82

+ IPMI05

19.6 accct0224
24.7 atacca253
35. 3 agactt163

 

 

64

9

Figure 3.3: QTL for day-neutrality detected in the RP progeny of ‘Tribute’ x ‘Honeoye
evaluated in St. Paul, Minnesota, 2005. This population was newly constructed for the
present study (see text for details). Four QTL were detected at the LOD threshold of 2.4
in linkage groups 7, 17, 20 and 28 closest to markers agtcag305, aggca1187, agacaa174

and agactc179 respectively.

 

 

 

 

 

 

lmLG7
o -s M on A or a) \I on co 8
0.0 ata/ctc365 I I I I I - 1 r L
6.0 atg/cg-118
11.1 agtlcagZZS
1
27.4 act/ctt117
37.9 agtlcag305 ;
z
O
S
52. 0 ag t/caa230

 

 

 

 

 

 

 

65

Figure 3.3 (Cont’d).

 

 

 

 

 

 

 

 

ImLG1 7

0. 0 egg/can 87 I

8. 1 ata/cac368
11.5 atg/011148
14.4 atg/cth48
24.5 ata/c6415
28.7 atg/cag205
41 .9 ata/c0167
47. 9 aag/ctg119

 

 

2'
1°»
M

O

-I
3

-8
~17
-9

99
pt
-8
-6

0L

 

 

 

 

 

66

Figure 3.3 (Cont’d).

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

ImLGZO
N on A on a» ‘1 on o 8
0.0 act/ctg193 Tr' . r r 1 . .
2.6 act/ctt340
5.2\_/aga/caa174 g
6.1/“Nact/ctg196 is
‘1"
(A)
12.7 agalcac153
15.2 atg/ctg160
lmLGZB
a N u A m a) V on o 8
0.0 agalctc179 1 1 I I 1 I 1 L
E
Z
O
‘1"
vA
21.0 aag/cag166 ° ””05
—O— MN05
—O— MN05
—O— MN05

 

 

 

67

9

Figure 3.4: QTL for day-neutrality detected in the RP progeny of ‘Tribute’ x ‘Honeoye
evaluated in Beltsville, Maryland, 2005. This population was newly constructed for the
present study (see text for details). Only one QTL was detected at the LOD threshold of

4.01 in linkage group 17 closest to marker aggcat187.

lmLG17

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

0.0 aggcat187 % o I ,
o
‘3‘
8.1 atam0368
11.5 atgctt148
14.4 atgct9248
+ RPMDO5
24.5 atactt415 1
28.7 atgngOS
41,9 atactt167
47,9 aagctg119

 

 

68

9

Figure 3.5: QTL for day-neutrality detected in the RP progeny of ‘Tribute’ x ‘Honeoye
evaluated in Corvallis, Oregon, 2005. This population was newly constructed for the
present study (see text for details). Two QTL were identiﬁed with a LOD score above

2.0, but they were below the 5% LOD threshold of 3.13.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

ImLG14
O _. to or) 35 U! or N co (D 8
0.0 agg/ct9217 O I I I I I I r r r
:0
O
1”
14.9— agtlcga112
16. 2 acalcag 143
19. 3 aa g/ca9257
22. 1 ag dctg1 78
23.6 ag glcga183
25. 1 ag t/cga320
38.0 agtlcat370 >
42. 8 ata/cct1 10 \
47.3 V agg/cga86

 

 

69

Figure 3.5 (Cont’d).

 

 

 

 

 

 

 

 

 

 

lmLG1 7

0. 0 ag glcat187

8. 1 ata/cac368
1 1 .5 atg/ch148
14.4 atg/ct9248
24. 5 ata/01141 5
28.7 atg/ca9205
41.9 ata/c111 67
47.9 aag/ctg119

#9080

-L

-

I-L
-9
-6

01

 

 

u.-€

 

 

 

70

 

 

+ OR05
+ OR05

 

 

Figure 3.6: QTL for day-neutrality detected in the RP progeny of ‘Tribute’ x ‘Honeoye’
evaluated in Watsonville, California, 2005. This population was newly constructed for
the present study (see text for details). Only one QTL was detected at the LOD threshold

of 3.3 in linkage group 7 closest to marker agtcag225.

 

 

 

 

 

 

 

 

 

 

lmLG7
O A N (A & 0| 0)

0.0 atactc365 I I I, I I
6.0 atgog-118

Ii
11.1 391089225 3;

b

O

I.”

+ RPCA05

27.4 actctt117
37.9 agtca9305
52.0 agtcea230

 

 

 

 

 

 

 

71

References

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