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BULK SEGREGANT ANALYSIS FOR BLOOM TIME
QTL IN SOUR CHERRY (Prunus cerasus L.)

presented by

Ann Marie Bond

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MS. degree in Plant Breedirg and Genetics/Horticulture

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6/01 c:/CIRC/DateOuetp65-p.15

BULK SEGREGANT ANALYSIS FOR BLOOM TINIE QTL 1N SOUR CHERRY
(Prunus caasus L.)

By

Ann Marie Bond

A THESIS
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
MASTER OF SCIENCE
Department of Horticulture and Genetics

2004

 

5 Li; , {{[Ellrllr I\ll..| fill IL. i’li

ABSTRACT

BULK SEGREGANT ANALYSIS FOR BLOOM TIME QTL IN SOUR CHERRY
(Prunus cerasus L.)

By Ann Marie Bond

Spring freeze damage to sour cherry (Prunus cerasus L.) flower buds is the major
limiting factor to sour cherry production in the US. Significant crop reductions from
spring freezes occur approximately every three years. In 2002, the losses to spring freeze
damage were particularly devastating with 95% of the sour cherry crop in Michigan
destroyed. One breeding approach to minimize the potential for freeze injury to sour
cherry flowers is to develop late-blooming cultivars that would have an increased chance
of avoiding spring freeze damage. Fortunately there is extensive variation for bloom time
in sour cherry with extremely late blooming gerrnplasm available.

The goal of this project was to investigate the inheritance of bloom time in
tetraploid sour cherry (2n = 4x = 32) using a QTL approach. The progeny population
used was from a cross between ‘Balaton’ and the late blooming sour cherry cultivar
‘Surefire’. QTL discovery was done using a bulk segregant approach comparing late and
early blooming progeny individuals using SSR and AFLP markers. Using this approach,
an AFLP marker was identified that was significantly linked to a putative QTL, termed
blm3, controlling bloom time. The late allele, contributed by the late blooming parent
‘Surefire’, explained 16.5% of the phenotype variance and delayed bloom time by
approximately 15 degree days. Future characterization of this QTL and other QTL
controlling bloom time may suggest ways to delay bloom in sour cherry and other

Rosaceous species, thereby reducing the probability of spring freeze damage and crop

loss.

DEDICATION

I would like to dedicate my thesis to the men and women of the United States Armed

Forces who unselfishly risk their lives, home and abroad, to preserve the way of life that I

have the liberty and opportunity to enjoy.

iii

ACKNOWLEDGEMENTS

I am very grateful to my advrsor, Dr. Amy Iezzoni, for her guidance, financial
support, and editorial assistance. I would like to thank my committee, Dr. Dechun Wang,
Dr. Jim Hancock, and Dr Dave Douches, for their patience, understanding, and last
minute advice. Also, I would like to extend countless thanks to members of both the
Iezzoni lab and the Hancock lab -— your wonderful sense of humor, words of advice, and

constant guidance were of immeasurable value.

To my roommates Lee Ann Pramuk and Janelle Glady - without whom I’d never
have gotten his far — thanks so much for strawberries and coconuts! I would also like to
thank the 7th Michigan Volunteer Infantry Company B for their constant support and love
— you have become my Michigan family and will forever have a special place in my

heart.
And finally, Mom, Dad, Sarah, and Seth — Thanks for all the constant support,

patience, and love. You have helped me attain the seemingly impossible by believing in

me when I needed it the most! Bluebirds and Dandelions to you always!

iv

TABLE OF CONTENTS

LIST OF TABLES
LIST OF FIGURES
I. INTRODUCTION

II. LITERATURE REVIEW.
A. Origin and history of sour cherry
B. Floral development and freeze susceptibility
C Variation 1n bloom time ...
D. Genetic analysis of bloom time in Prunus ...

III. MATERIALS AND METHODS
A. Plant material
B. Evaluation of bloom time . .

C. Determination of early and late blooming bulk populations

D. DNA isolation
E. SSR analysis
F. AFLP analysis
G. QTL analysis

IV. RESULTS
A. Variation 1n bloom time
B. Selection of early and late blooming bulk populations
C. SSRanalyses
D. AFLP analyses

E. QTL analysis of marker E-AAA/CGT265 with bloom time

V. DISCUSSION

VII. APPENDICES ..
A. Abbreviated SAS input files
B. Population data for 1999, 2000, 2001
C. Bulk selection
D. Degree Day data
E. SSR raw data
F. AFLP raw data
G. Statistical comparisons

VI. REFERENCES

macaw;

10
10
12
12

14
16
17
20

25

33
34
36
37

47
48

vii

14

29

33

49

LIST OF TABLES

Table l. Prunus linkage group(s) and reference information for the twenty-eight SSR
primer pairs used in this study - Linkage groups are numbered as in Joobeur et al.
(2000)
... ... 11

Table 2. Selective nucleotide sequences used for AFLP analyses with the restriction
enzymes Eco RI and Mse I.
13

Table 3. Bloom data code and degree days (°C) to reach 50% bloom for the nine progeny
selected for the early bulk ( = no data, letters refer to bloom code as described in

Materials and Methods).
. .. . .. . .. . . 16

Table 4. Bloom data code and degree days (°C) to reach 50% bloom for the nine progeny
selected for the late bulk (- = no data, letters refer to bloom code as described in
Materials and Methods).
... 17

Table 5. SSR primer pairs, fragments observed, and percent polymorphic fragments for
Balaton, Surefire, and the 18 individuals in the early and late bloom bulk
populations" (see Appendix F for SSR genotyping data).

Table 6. AFLP analysis of Balaton, Surefire, and the bulk progeny individuals utilizing
primers Eco RI +ANN and Mse I + CNN.
.. . 21

Table 7. AFLP analysis of Balaton, Surefire, and the bulk progeny individuals utilizing
primers Eco RI +AN and Mse 1+ CNN.
Table 8. Presence or absence of E-AAA/M-CGT265 in Balaton, Surefire and the bulk

progeny individuals. (na = no amplification in this particular PCR reaction).

Table 9. Chi Square Analysis of the segregation of marker E-AAA/M-CGT265 in the

Balaton and Surefire population to a 1:1 and 1:2. (*Significance at P < 0.05 for
deviation from expected ratio, as = not significant).

25

vi

LIST OF TABLES (continued)

Table 10. Analysis of variance for the association of marker state for E-AAA/M-

CGT265 with heat unit accumulation to 50% bloom time among 151 progeny
evaluated in 2002 and 2003.
27

Table l 1. Mean heat unit accumulation for 50% bloom for progeny separated by the
presence or or absence of blm3. (a and b denote significant difference within
columns at P < 0.05). '

27

APPENDIX

C. Table 1. Day and degree day comparisons for 50% bloom to identify early and late
blooming individuals for the early and late bulks (- = no assignment).

D. Table 1. Growing degree days data (°C) for all Balaton x Surefire progeny individuals.

E. Table 1. Presence or absence data for tested SSR pairs including parents and progeny
evaluated.

44

F. Table 1. Presence or absence for E-AAA/M-CGT265 fi'om all the parents and progeny
evaluated.
47

G. Table 1. SAS output for “group” comparison where group a is absent for E-AAA/M-
CGT265 and b is present (therefore representing blm3 and Blm3 respectively,
includes 2002 and 2003).
48

vii

LIST OF FIGURES

Figure 1. Frequency distributions of 50% bloom for the progeny population in 2002 (A)
and 2003 (B). Bloom dates for the parents Balaton and Surefire are identified by
B and S, respectively. Degree days were calculated in Celsius with a base temp of
4.4°C.
15

Figure 2. Frequency distributions of the degree days needed for the progeny individuals
to reach 50% bloom in 2002 (A) and 2003 (B). The degree days for the parents
Balaton (B) and Surefire (S) are shown as inserted letters respectively. White bars
indicate degree days for progeny within the early bulk and dark grey bars for the
respective late bulk.
... 18

Figure 3. Segregation for E-AAA/M-CGT265 among the parents and bulk progeny in this
particular PCR reaction. B= Balation, S=Surefire, Early bulk = 2(6), 2(61), 3(2),
3(24), 3(25), 3(46), 4(31), 4(34), Late bulk = 2(7), 3(26), 3(51), 4(22), 4(24),
4(45).
24

APPENDIX

B. Figure 1. Frequency distributions of 50% Bloom for the Balaton x Surefire progeny
population over the first 3 years of bloom: 1999 (A), 2000 (B), and 2001 (C).
Bloom date for the parents Balaton (B) and Surefire (S) are shown as inserted
letters correspondingly.

34

viii

I. INTRODUCTION

Spring freeze damage to sour cherry (Prunus cerasus L., 2n = 4x) flower buds is
the major limiting factor to sour cherry production in the United States. (Ricks, 1992).
Significant crop reductions from spring freeze events occur approximately every three
years. In 2002, the losses to spring freeze damage were particularly devastating with
~95% of the Michigan sour cherry crop destroyed (Kleweno and Matthews, 2003). The
genetic uniformity of sour cherry production in the U. S., a monoculture of the 400 year
old cultivar Montmorency, also set the stage for such a disaster.

Within the flower, the pistil is the most sensitive tissue, and in the vast majority of
freeze events, the pistil tissue is killed while the other flower tissues remain un-injured
(Dennis and Howell, 1974). For example, sour cheny leaves can cold acclimate to
survive temperatures of -13 2°C, where as cherry pistils are killed at temperatures
between -2 to -3°C (Owens et al., 2001). Initial investigations suggest that cherry pistils
may be deficient in their ability to cold acclimate due to absence in CBFI activity
(Owens et al., 2002). CBFI is a transcription factor that has been demonstrated to be
required for cold acclimation in other plant species (Jaglo et al., 2001).

One breeding approach to minimize the potential for freeze injury to sour cherry
flowers is to develop late-blooming cultivars. Since flowers become increasingly
sensitive to freeze injury as they develop in the spring (Ballard et al., 1971), late
blooming cultivars have an increased chance of avoiding spring freeze damage.

Fortunately there is extensive variation in sour cherry for bloom time, as well as available

gerrnplasm that blooms significantly later than Montmorency (Iezzoni and Hamilton,
1985; Iezzoni and Mulinix, 1992).

Previous studies in Prunus species identified Quantitative Trait Loci (QTL) for
bloom time. In an inter-specific peach x almond cross, a major QTL for bloom time
(called Lb) was identified on linkage group 4 (Arus et al., 1999; Ballester et al., 1998). In
a peach cross, 6 QTL for flowering time were identified with a large QTL on Group S
(Yamamoto et al., 2001). In the sour cherry cross Rheinische Schattenmorelle x Erdi
Boterrno, two QTL for bloom time were identified (Wang et al., 2000). However, the
alleles identified at these loci, blml and blm2, conferred early bloom. A second cherry
cross, Balaton x Surefire, was made to develop a pseudo-testcross population for the
identification of late blooming QTL allele. Surefire was chosen as a parent because it is
one of the latest blooming sour cherry selections.

Since bloom time in sour cherry has a high heritability (Wang et al., 2000) and
QTL analyses have been successfirl in Prunus, we hypothesize that it should be possible
to identify additional or orthologous QTL for bloom time in the Balaton x Surefire cross.
Our objective was to identify QTL controlling bloom time and the alleles at these QTL
that contribute to late bloom. QTL discovery was done using a bulk segregant approach
(Mikas et al., 1996; Michelrnore et al., 1991) comparing late and early blooming progeny
individuals with_Simple Sequence Repeat (SSR) and Amplified Fragment Length

Polymorphism (AFLP) marker analysis.

 

 

 

II. LITERATURE REVIEW

A. Origin and histog of sour chem

Cherries are members of the Rosaceae family, which as a whole, is economically
important in the globe’s temperate regions for both their fi'uits and ornamentals. Cherries
have been valued since ancient times as one of the first tree fi'uits to ripen in temperate
growing regions. The two main groups of cherries, the diploid sweet (Prunus avium, 2n
= 2x = 16) and tetraploid sour cherries (syn. tart cherries, 2n = 4x = 32), are reported to
have originated in an area that includes Asia minor, Iran, Iraq, and Syria (Watkins, 1976).
The first diploid Prunus species arose in central Asia: sweet, sour, and ground cherry
Prunusfi-uricosa, (a low growing bush cherry native to Russia) were early derivatives of
this ancestral Prunus. Sour cherry is believed to have arisen through natural

hybridizations between ground cherry and sweet cherry (Olden and Nybom, 1968).

B. FIoLaLdevelopment and freeze susceptibilig

Cheny production is limited to temperate regions that experience moderately cold
winter temperatures. The cherry tree requires a dormancy period each year that begins
with defoliation in the fall. Spring growth starts when the dormancy or chilling
requirement has been satisfied and temperatures increase sufiiciently in the spring. In
colder regions, cherry production is limited by cold mid-winter temperatures. For
example, temperatures below -3 0°C can result in wood or flower bud injury, particularly
in the more cold-sensitive sweet cherries. However, in some chen'y growing regions,

low-temperature damage to flower buds is the most important factor limiting yields.

Vegetative bud burst and flowering occur in the spring in response to warm
temperatures favorable for rapid plant growth (above 10°C). Individual cherry flowers
are perfect, consisting of sepals, petals, stamens, and a pistil. The stamen is made up of a
filament and a pollen-bearing anther where as the pistil is comprised of the stigmatic
surface, the style and the ovary containing a pair of ovules. In Prunus, one ovule typically
develops into the seed while the other ovule aborts very early. The ovary develops into
the fleshy pericarp of the drupe fruit.

Since flowers become increasingly sensitive to freeze injury as they develop in
the spring (Ballard et al., 1971; Dennis and Howell, 1974), late blooming cultivars have
an increased chance of avoiding spring fi'eeze damage. Therefore, late bloom is a
common goal in Prunus breeding programs (Bailey and Hough, 1975).

Difi‘erences in bloom time among selections are fi’equently compared using heat
accumulation above a given threshold to reach a designated pheonological state
(Baskerville, 1968; Baskerville and Emin, 1969). The resulting calculated “degree-days”
is therefore a more usefirl comparative measure for bloom time than calendar days. Since
all the flower buds on a tree do not open at the same time, bloom time is sometimes

evaluated when 50 percent of the blossoms are estimated to be open, is. 50% bloom

(Wang et al., 2000).

C. Variation in bloom time
Sour cherries offers a unique opportunity to study late bloom in Prunus, as sour
cherry and its parental species, P. fruticosa, are the latest blooming commercially

important Prunus species (Iezzoni et al., 1990). Presumably P. fi-uticosa evolved

extremely late bloom time to avoid spring freeze damage in its native habitat in central
Russia. Due to continued introgression between sour cherry and P. fruticosa, there is
extensive variation in sour cherry for bloom time, including germplasm that blooms
significantly later than Montmorency (Iezzoni and Hamilton, 1985; Iezzoni and Mulinix,

1992)

D. Genetic analysis of bloom time in Prunus

Various linkage maps have been created in Prunus species; however, most of the
linkage maps have been constructed from diploid crosses among peach selections or
between peach and almond (Arus et al., 2003; Ballester et al., 1998; Yamamoto et al.,
2001). Recently, SSRs (syn. micro satellite markers) have been suggested to be a
valuable marker resource for comparative mapping (Aranzana et al., 2001). For example,

109 SSRs that were originally developed for peach and cherry, are being mapped on an

almond x peach F2 population. They tested the marker variability between 25 cultivars

(14 peach and 11 nectarine) and found 24 of the 35 SSRs to be polymorphic and
consequently could distinguish between all of the individuals in the test (Aranzana et al.,
2001). Testolin (2000) did similar research studying genetic origin and fingerprinting in
peach, and all were polymorphic. Of the 26 SSR primer pairs they tested, 17 exhibited
Mendelian inheritance. SSRs fiom peach and cherry were also found to be useful to
distinguish among sour cherry germplasm accessions (Cantini et al., 2001). Additionally,
AFLPs have been used to increase marker coverage and gene tagging.

Unfortunately publication of linkage maps from different researchers originally

resulted in different numbers associated with corresponding linkage groups. For example,

numbered consecutively, the nomenclature used by European researchers for
chromosomes 1, 2, 3, 4, 5, 6, 7, and 8 (Aranzana et al., 2003), are numbered 5, 2, 4, 6, 8,
3, 1, and 3, in a Japanese peach map [Groups 8 and 6 are shown as one linkage group (3)]
(Yamamoto et al., 2001). This complicates the comparative QTL analysis. Fortunately,
since 2002, the nomenclature of the European groups, as first published by J oobeur et al.
(2000), has been adopted as the oflicial nomenclature for Prunus. -

Despite the preliminary stages of linkage mapping in diploid Prunus, QTL for
bloom time have been reported independently by three groups (Ballester et al., 1998;
Yamamoto et al., 2001; Wang et al., 2000). In an inter-specific peach x almond cross, a
major QTL for bloom time (Lb) was identified on linkage group 4 (Arus et al., 1999;
Ballester et al., 1998). In a peach cross, 6 QTL for flowering time were identified with a
large QTL on the Japanese peach map, Group S (Yamamoto et al., 2001). In the sour
cherry cross Rheinische Schattenmorelle x Erdi Botermo, two bloom time QTL were
identified (Wang et al., 2000); however, the alleles blml and blm2 conferred early bloom.
The objective of this study is to identify QTL controlling bloom time, but also to identify
the alleles at these loci that may contribute the desired late bloom phenotype.

Because bloom time in sour cherry has a high heritability (Wang et al., 2000) and
QTL analyses have been successful in Prunus, we hypothesize it should be possible to
identify additional or orthologous QTL for bloom time in a second sour cherry cross.
This second cherry cross, Balaton x Surefire, was designed to assist in the identification
of late blooming alleles as Surefire is one of the latest blooming sour cherry selections.
Marker analyses of the progeny of this cross would initially consist of markers previously

been demonstrated to be linked to published bloom time QTL (Arus, 2003; Bliss et al.,

2002; Wang et al., 1998; Yamamoto et al., 2001). Most of these markers are SSRs.
Since these markers have been previously mapped in other Prunus species, it should be
possible to determine if marker order and distance have been conserved between sour
cherry and the other Prunus species. Ifnecessary, genome coverage to look for
additional QTL could be accomplished with AFLP analyses as was performed in peach to
map a gene associated with nematode resistance (Lu et al., 1998). -

QTL identification in a heterozygous polyploid crop such as sour cherry is more
challenging than in a diploid. Only markers that meet the criteria of a single dose
restriction fragment can be placed on the linkage map, and most loci are duplicated in a
polyploid. This requires that only markers in simplex in one or both parents can be
mapped. QTL alleles must also meet this criteria for linkage mapping.

Because identifying QTL in a polyploid species can be quite a challenge, the
strategy is to use a bulk segregant analysis (Michelmore et al., 1991), whereby the initial
goal is to identify marker polymorphisms that differ between a group of late blooming
and early blooming selections. Bulk segregant analysis has previously been used to
identify markers associated with QTL for quantitative traits (Mansur et al., 1993; Mikas
et al., 1996). The association of marker(s) with a QTL for bloom time would

subsequently be tested by scoring all progeny for the marker of interest.

1]]. MATERIALS AND METHODS

A. Plant material.

The two sour cherry cultivars used as parents to produce the population for QTL
analysis were ‘Balaton’ and ‘Surefire’. Balaton is a landrace Hungarian variety called
Ujfehertoi Furtos. Surefire is a cultivar released from the New York State Agricultural
Experiment Station, Cornell University, from a cross between Borchert Black Sour x NY
6935 (Richmorency x Schattenmorelle) (R. Andersen, pers. comm.) Surefire was chosen
as the late blooming parent as it is one of the extremes for this trait. The Balaton x
Surefire cross was made in 1996. One hundred ninety-seven (197) seedlings were
planted at the Michigan State University Clarksville Horticultural Experimental Station
(CHES), Clarksville, Michigan in the spring of 1998. The seedlings were planted in field
number 27 e and spaced within a row 1.5 meters apart, with the rows 6.1 meters apart.
The following progeny individuals were not included in this study as they are suspected
to have resulted fi'om out-crossing or self-pollination [designated by row(tree)]: 1(65),
2(20), 2(34), 2(54), 3(06), 3(11), 3(35), 4(08), 4(23), 4(36), 4(39), 4(47) (A. Sebolt and
N. Hauck, pers. comm.) With these individuals eliminated, the final population size was
185. The following progeny have not yet been genotyped for their S-alleles: 2(5), 2(10),
2(14), 2(21), 2(22), 2(28), 2(29), 2(30), 2(31), 2(46), 2(49), 2(60), 3(9), 3(10), 3(13),
3(23), 3(32), 3(43), 3(50), 3(51), 3(54), 3(56), 3(60), 4(1), 4(4), 4(5), 4(17), 4(18), 4(19),
4(20), 4(30), 4(33), 4(3 7), 4(50), 4(51), 4(52), 4(53), 4(54), 4(59), 4(60), 4(63), 4(64),

4(65), and 4(66).

B. Evaluation of bloom time.

Bloom time observations were made daily (at 10AM beginning mid April) to
record the date when approximately 50% of the flowers had opened for each seedling
tree. Time to bloom was expressed as degree days (DD) from January 1 with a base
temperature of 4.4°C using hourly temperature readings collected at the Automated
Weather Station located at CHES (Baskerville and Emin, 1969). Daily heat unit
accumulation was calculated by summing the positive differences of hourly temperature
readings minus 4.4°C, then dividing by 24. On the date of 50% bloom, heat unit
accumulation was calculated to hour 10, the approximate time data was recorded. Bloom

time was evaluated over five years (1999 - 2003).

C. Determination of early and late blooming bulk populations.

In order to divide the population into two extreme phenotypic groups to form the
early and late bulks, the progeny were organized chronologically for each year using the
date of 50% bloom for each individual. For example, in 2003, the earliest blooming
progeny [individuals 3(24) and 3(25)] reached 50% bloom on April 28tll or 177.2 DD.
This day was recorded as day “a”. April 2911. was recorded as day “b” and so on until day
“c”. This was performed for each year of available bloom data. A table was made
including alist of progeny with their bloom date for each year and the corresponding
bloom day letter code. Progeny with consistent early blooming dates and late blooming
dates were chosen to form the “early bulk” and “late bulk”, respectively. The individuals

in the early and late bulks were screened using the SSR and AFLP markers described

below. Any marker(s) that was found to be present in one bulk but absent in the other,

was subsequently genotyped for the entire progeny population of 151.

D. DNA isolation:

Young leaves were collected from the parents and each progeny individual,
placed immediately on dry ice, transported back to the laboratory, and placed in the -80°C
freezer for 24 hours. The samples were then fi'eeze-dried for at least 48 hours and then
frozen at -20°C for storage. DNA was isolated using the CTAB method described by

Stockinger et al. (1996).

E. SSR analysis:

Twenty-eight SSR primer pairs from both cherry and peach with known Prunus
linkage map positions were used to screen the parents and bulk progeny individuals
(Aranzana et al., 2001; Cantini et al., 2001; Cipriani et al., 1999; Dirlewanger et al.,
2002; Sosinksi et al., 2000; Testolin et al., 2000; Wunsch et al., 2002) (Table 1). In an
attempt to target those regions where bloom time QTL had previously been reported in
Prunus, 10, 5, and 3 SSR primer pairs that map to Prunus linkage groups 1, 4, and 2,
were tested (respectively). Primer pairs were selected by proximity (within 20 cM) to the
published QTL. PCR amplifications were performed in a Perkin Elmer Cetus DNA
Thermocycler 480 following procedures described in Cantini et al. (2001). To test the
success of the PCR reaction, PCR products were separated by electrophoresis in 1%
agarose gels with 0.1% ethidium bromide. For genotyping, the PCR products were mixed

with 7.6 ul of fonnamide dye (10 ml 98% forrnamide, 200 ul 0.5mM EDTA, 10 mg

10

Table 1. Prunus linkage group(s) and reference information for the twenty-eight SSR
primer pairs used in this study — Linkage groups are numbered as in Joobeur et al. (2000).

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Primer Pair Linkage Group (5) Reference

BPPCT008 G6 Dirlewanger et al. (2002)
BPPCTOI 5 G4 Dirlewanger et al. (2002)
BPPCT021 Gl Dirlewanger et al. (2002)
BPPCT023 G4 Dirlewanger et al. (2002)
BPPCT038 G5 Dirlewanger et al. (2002)
BPPCT04O G4 Dirlewanger et al. (2002)
CPPCT002 G3 Aranzana et al. (2001)
CPPCT003 G1 and G4 Aranzana et al. (2001)
CPPCT026 G1 Aranzana et al. (2001)
CPPCT027 G1 Aranzana et al. (2001)
CPPCTO34 G1 Aranzana et al. (2001)
PceGA25 G5 Cantini et a1. (2001)
PceGA59 G1 Cantini et al. (2001)
PceGA34 G2 A. Iezzoni (pers. comm.)
pchgms3 G1 Sosinski et al. (2000)y
pchgmsS G6 Sosinski et al. (2000)y
PMSZ G7 Cantini et al. (2001)
PMS3 G4 Cantini et al. (2001)
PMS67 G1 Cantini et al. (2001)
UDP96—008 G3 Cipriani et al. £999) 2
UDP96-018 G1 Cipriani (1999z
UDP97-403 G3 Cipriani (1999) z
UDP98-022 G1 Testolin et al. (2000) y
UDP98-025 G2 Testolin et al. (2000)y
UDP98-405 G7 Cipriani (1999)z
UDP98-409 G8 @riani (1999)z
UDP98-411 G2 Testolin et al. (2000) y
UDP98-412 G6 Testolin et al. (2000) ’

 

 

 

2 Further used in Prunus persica and P. avium by Testolin et a1. (2000),
Wunsch et al. (2002).

y Further used in P. persica L. and P. avium L. by Wunsch et al. (2002).

11

 

bromophenol blue, 10 mg Xylene Cyanol), heated at 95°C for 5 minutes and immediately
placed on ice. Five microliters of each sample were loaded on a denaturing 6%
polyacylamide gel. The samples were then electrophoresed at 80 watts for 2.5 hours and
stained using the Silver Staining Protocol from the Promega Technical Manual. (Promega

Corporation; Madison, Wisconsin)

F. AFLP analysis:

DNA digestion, adaptor ligation, pre-selective amplification and selective
amplifications were carried out according to standard procedures described by Vos et al.
(1995). Primer combinations included three selective bases for one primer (Mse I) and
both two and three selective bases for the other (Eco RI) (Table 2). PCR products were
first checked for successful amplification by electrophoresis in 1% agarose gels stained
with 0.1% ethidium bromide. The PCR products were then prepared for polyacrylamide
gel electrophoresis by mixing the reaction products with 7.6 ul of formamide dye (10 ml
98% formamide, 200 111 0.5mM EDTA, 10 mg bromophenol blue, 10 mg Xylene
Cyanol), heated at 95°C for 5 minutes and immediately placed on ice. Five microliters of
each sample were loaded on a denaturing 6% polyacrylamide gel. The samples were then
electrophoresed at 80 watts for 2.5 hours and stained using the silver staining protocol

according to Promega (Promega Corporation; Madison, Wisconsin).

G. QTL analysis:
A Nested Factor Design was used for a Single Marker QTL Analysis to determine

if any marker is associated with bloom time. The linear additive model for the Analysis

12

of Variance is presented below. The two years represented the replications for this model.
Computations were done using SAS (SAS Institute, Inc., 1999). Input files can be found

in Appendix A.

Yijkl = 11 + 8T00P1+ yearj + tredgroum + year’SIOUPij + $in
where Yijk] = date of bloom in degree days
11 = grand mean _

group; = marker present/absent by genotype
yearj = replications, 2002 and 2003
tree(group)k = progeny nested within the group
year*groupij = interaction

$in = years

Table 2. Selective nucleotide sequences used for AFLP analyses with the restriction
enzymes Eco RI and Mse I.

 

Two base pairs Three base pairs
EcoRI+AA/MseI+CAA EcoRI+AAA/MseI+CGT
EcoRI+AA/MseI+CAC EcoRI+AGG/MseI+CAC
EcoRI+AC/MseI+CAA EcoRI+ATA/MseI+CAC
EcoRI+AC/MseI+CAC EcoRI+ATA/MseI+CCG
EcoRI+AC/MseI+CAG EcoRI+ATA/MseI+CCT
EcoRI+AG/MseI+CAA
EcoRI+AT/MseI+CAC
EcoRI+AG/MseI+CAC
EcoRI+AT/MseI+CAA
EcoRI+AT/MseI+CAC

 

 

 

 

 

 

 

 

 

 

 

 

 

 

13

IV. RESULTS

Maligtion in bloom time

Bloom time was recorded for all blooming progeny in the Balaton x Surefire
population over five years (A. Iezzoni, unpublished data) (Appendix E). The number of
blooming progeny reached a maximum of 175 individuals in 2002. In 1999, only 33 of
the 3 year old seedlings bloomed followed by 85 individuals in 2000, 143 individuals in
2001, 175 individuals in 2002 and finally, 170 individuals in 2003. This trend likely
represents the differences in the length of juvenility period among the seedlings with all
the seedlings flowering by age seven. To maximize the number of progeny evaluated -
only year 2002 and 2003 bloom data was used in the Single Marker Analysis (Appendix
A for SAS input code).

Bloom data was converted fi'om calendar days to degree days in order to compare
bloom time over years on a heat unit basis. The progeny distribution for bloom time
spanned 90.9 heat units in 2002 and 109.1 heat units in 2003 (Figure 1). The heat units
accumulated for 50% bloom differed significantly between the two years; however, the
year x progeny interaction was not significantly different over the two years. Balaton
reached 50% bloom earlier than Surefire, exhibiting differences of 44.5 degree days in
2002 and 40.9 degree days in 2003.

The progeny exhibited transgressive segregation for both early and late bloom
time (Figure 1). In 2002, 28% of the progeny bloomed earlier than the early blooming
Balaton parent and 5% of the progeny bloomed later than the late blooming Surefire

parent. In 2003, 34% of the progeny bloomed earlier than Balaton and 6% of the progeny

l4

 

2002 Bloom Distribution

 

 

 

 

 

 

 

 

Number of Trees In Bloom

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Number of Trees In Bloom

 

 

 

 

 

 

Figure 1. Frequency distributions of 50% bloom for the progeny population in 2002 (A)
and 2003 (B). Bloom dates for the parents Balaton and Surefire are identified by B and
S, respectively. Degree days were calculated in Celsius with a base temp of 4.4°C.

15

 

bloomed later than Surefire. These early and late blooming progeny individuals provided

the opportunity to select individuals for bulk segregant analysis.

B. Selection of early and late blooming bulk populations

In order to divide the population into two extreme phenotypic groups to form the
early and late bulks, the progeny were organized chronologically for each year using the
date of 50% bloom for each individual. The population was divided into two extreme
groups (denoted early and late) and the bloom data was organized chronologically for
each year, the date of 50% bloom for each individual. Nine progeny with consistent early
blooming dates were chosen to be a part of the “early bulk” for marker analysis: 2(6),
2(61), 3(2), 3(24), 3(25), 3(46), 3(54), 4(31), and 4(34) (Table 3). The range in heat units
to reach 50% bloom of the individuals chosen for the early bulk was 177.2 to 216.6
degree days in 2003. The progeny population mean was 232.9 degree days. These
selected progeny had noticeably earlier bloom time in comparison to the population mean
over all years.
Table 3. Bloom data code and degree days (°C) to reach 50% bloom for the nine progeny

selected for the early bulk (— = no data, letters refer to bloom code as described in
Materials and Methods).

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Early bulk 1999 2000 2001 2002 2003

rank hours Rank hours rank hours rank hours rank hours
2(6) f 226.7 c 221.8 b 188.7 - 199.7 - 211.2
2(61) a 187.2 c 221.8 b 188.7 a 179.8 c 195.2
3(2) - - c 221.8 b 188.7 f 193.3 - 216.6
3(24) b 192.4 a 211.8 b 188.7 c 189.1 a 177.2
3(25) - - - - - - f 193.3 a 177.2
3(46) - - - - b 188.7 c 189.1 b 186.9
3(54) - - - - c 205.3 c 189.1 e 208.1
4(31) - - e 234.5 a 178.7 c 189.1 c 195.2
4(34) - - (1 228.1 b 188.7 f 193.3 a 177.2

 

l6

 

Similarly for late bloom, the last day of 50% bloom for the population in 2003

was May 13th or 286.3 DD. This day was recorded as day “2”, May 12th was recorded as

“y” and so on until day “v”. Nine progeny with consistent late blooming dates were

chosen to be part of the “late bulk” for marker analysis: 2(7), 2(39), 3(26), 3(51), 4(22),

4(24), 4(32), 4(45), and 4(66) (Table 4). The nine progeny individuals selected for each

bulks represented 4.9% of the total population. The range in heat units to reach 50%

bloom of the individuals chosen for the late bulk was 246.6 to 286.3 degree days in 2003.

The progeny population mean was 232.9 degree days.

Table 4. Bloom data code and degree days (°C) to reach 50% bloom for the nine progeny
selected for the late bulk (- = no data, letters refer to bloom code as described in Materials

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

and Methods).
Late bulk 1999 2000 2001 2002 2003

rank hours Rank hours rank hours rank hours rank hours
2(7) v 284.5 w 289.7 - 239.3 x 263.3 w 266.6
2(39) - - 2 343.4 2 274.8 y 268.0 y 283.6
3(26) - - - - - - - - 2 286.3
3(51) - - - - x 255.9 y 268.0 - 246.6
4(22) 2 318.8 2 343.4 x 255.9 w 257.8 2 286.3
4(24) - - y 324 1 2 274.8 2 270.7 2 286.3
4(32) - - - - - 221.8 w 257.8 w 266.6
4(45) - - x 306.6 x 255.9 v 251.1 x 280.6
4(66) - - x 306.6 - - y 268.0 2 286.3

 

C. SSR analyses:

The SSR genotypes for 28 primer pairs were determined for the two parents and,

individually, for the 17 progeny in the early and late bulks. No fragments were amplified

with five of the primer pairs (Table 5). Considering the remaining primer pairs tested,

the number of fragments amplified from these sour cherry individuals ranged from 0 to 9

17

 

 

 

2002 Bloom Distribution

 

 

 

 

 

         

 

 

      

 

 

 

 

  
  

    

    

  

   

   
  

 

 

 

    
   

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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18

Degree Days at 50% Bloom

 

 

 

 

Figure 2. Frequency distributions of the degree days needed for the progeny individuals
to reach 50% bloom in 2002 (A) and 2003 (B). The degree days for the parents Balaton
(B) and Surefire (S) are shown as inserted letters respectively. White bars indicate degree
days for progeny within the early bulk and dark grey bars for the respective late bulk.

No-35: as. «225.: .8888 .8882 2852:: can 85E .2 8.53.. a; 82823.5 8...

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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19

(Table 5). Nine SSR primer pairs produced only monomorphic fragments, while the other
14 primer pairs amplified fragments were polymorphic among the parents and progeny
tested (Appendix F). None of the polymorphic SSR fragments were consistently present
in individuals from one bulk and absent from individuals in the other bulk. Therefore, this
SSR analysis did not reveal a marker that could be tested as a candidate for linkage to a

bloom time QTL.

D. AFLP analyses:

Fifteen AFLP primer combinations were used to screen the parents and
individuals from both the early and late bulks. All fragment sizes were scored for all the
individuals tested. The monomorphic and polymorphic fragments were summarized to
evaluate the feasibility of firture AFLP linkage mapping in this population (Table 6 and
7). Initially, primer combinations with three selective nucleotides for both Eco RI and
Mse I were used, which exhibited an average of 21 bands per gel (averaging 30%
polymorphism). Based upon Vilanova et al. (2003), two selective nucleotides were used
with the Eco RI primer producing on average of 39 bands per gel (averaging 14%
polymorphism).

Only one fragment was found that was present in one parent, absent in the other
parent and differentially present in the bulk progeny. The candidate marker of ~265 bp

was identified with AFLP primers Eco R1 + AAA and Mse I + CGT, and therefore the

marker name assigned to this fragment was E-AAA/M-CGT255 (Figure 3). This primer

pair was initially screened with both parents, eight early bulk individuals, and six late

20

 

 

 

 

 

 

 

 

 

 

 

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23

Figure 3. Segregation for E-AAA/M-CGT255 among the parents and bulk progeny in
this particular PCR reaction. B= Balation, S=Surefire, Early bulk = 2(6), 2(61), 3(2),
3(24), 3(25), 3(46), 4(31), 4(34), Late bulk = 2(7), 3(26), 3(51), 4(22), 4(24), 4(45).

    

AA AAA A
'1; AW 1-1 N V
M l‘ N m N N
vvvv vvv
VVI’ N n n V V

   

A
\O
v
N

A AAA
I-l AV '0 \O
\D NN N V
vvvvv
N MM 0") ('5

       

Table 8. Presence or absence of E-AAA/M-CGT265 in Balaton, Surefire and the bulk
progeny individuals. (na = no amplification in this particular PCR reaction).

I Balaton I absent I Surefire I presentJ

 

24

bulk individuals. E-AAA/M-CGT255 was present in Surefire, absent in Balaton and

present in all but one of the progeny in the late bulk and absent in all of the progeny in
the early bulk (Figure 3, Table 8). The one late bulk progeny individual that did not have
this marker, 4(45), was early in bloom time relative to the other individuals in the late
bulk. (Although in 2003, individual 4(45) bloomed 280.6 DD designated day “xT’ for that
year — Appendix C).

The differential presence of E-AAA/M-CGT265 between the parents and bulks

suggested that it might be linked to a genomic region important in the genetic control of
bloom time. Therefore this marker was fiirther screened on a larger number of progeny

individuals to test this association.

E. QTL analysis of marker E-AAA/M-CGTQQ with bloom time.

An additional 137 progeny were genotyped for marker E-AAA/M-CGT255

(Appendix F). The marker was present in Surefire, absent in Balaton and present and
absent in 45 and 106 progeny individuals, respectively (Appendix G). The marker was
tested for a 1:1 (+/-) ratio, the ratio expected from segregation of a single dose restriction
fragment. The marker segregation deviated significantly for this expected ratio, therefore

a 1:2 (+/-) ratio was tested. The segregation data fit a 1:2 (+/-) ratio. This suggested that

the segregation exhibited by marker E-AAA/M-CGT255 is due to significant skewing in

favor of the absence of the marker. It appears to show selection against the presence of

25

E-AAA/M-CGT265, which would mean the allele inherited fi'om Surefire is linked to

something being selected against.

Table 9. Chi Square Analysis of the segregation of marker E-AAA/M-CGT255 in the
Balaton and Surefire population to a 1:1 and 1:2. (*Significance at P < 0.05 for deviation
from expected ratio, n.s. = not significant).

 

 

Expected ratio ( + : -) Observed ratio ( + : - ) x:z
1 : 1 45: 106 23.4“
1 22 45 I 106 0.6 M’

 

 

 

 

 

The association of marker state for E-AAA/M-CGT265 and bloom time was tested

using Single Marker Analysis of Variance. Three analyses were performed, one for year

2002, one for year 2003, and then a third analysis with both years combined. In all three

analyses, marker E-AAA/M-CGT255 was significantly associated with bloom time

indicating linkage to a QTL controlling bloom time (Table 11). Specifically, the data

indicates the marker E-AAA/M-CGT265 is linked in coupling with a QTL allele in

Surefire that confers late bloom time. This putative QTL is named bIm3 following the
nomenclature of Wang et al. (2000) in which two QTL for bloom time (blml and bIm 2)
were identified.

When looking at the significance of growing degree days, both 2002 and 2003
show a significant difference between the two marker ‘groups’. In both cases, the present
fragment group, or BIm3 group is significantly later. When the degree day data was
analyzed over both years, again the two groups were significantly different which
prevents the possibility of combining the data. The allele BIm3 was again found to be

significantly associated with late bloom (Appendix H).

26

 

Table 10. Analysis of variance for the association of marker state for E-AAA/M-

CGT265 with heat unit accumulation to 50% bloom time among 151 progeny evaluated in
2002 and 2003.

 

 

 

 

 

 

Source df Mean Square P value P value
group 1 14277.00 18.88 <.0001
year 1 7860.37 69.19 <.0001
grorgf'year 1 135.30 1.19 0.2769
tree(group) 1 50 787 .79 6.93 <.0001
error 145 113.60

 

 

 

 

 

 

 

The mean heat unit accumulation to 50% bloom for those progeny that had the
late blooming BIm3 allele was significantly greater than the heat unit accumulation to
50% bloom for those progeny not exhibiting BIm3 and therefore by default, having the
bIm3 allele (Table 9). This QTL was associated with a delay in bloom time of 16.7 heat
units in 2002, 14.1 heat units in 2003, and 15 heat units in both years combined (Table
11).

Table 1 1. Mean heat unit accumulation for 50% bloom for progeny separated by the

presence or or absence of blm3. (a and b denote significant difference within columns at
P < 0.05).

 

 

 

 

 

 

 

Marker 2002 2003 2002 & 2003
BIm3 present 230.25 a 240.64 a 235.45 a
blm3 absent 213.52 b 226.54 b 220.03 b

 

 

Using the mean squares calculated in the Single Marker Analysis (Table 10), the
broad sense heritability for bloom time, and therefore the percent phenotypic variance

explained by this marker, was 16.5 %. This was calculated using the procedure outlined

below:

27

 

Source

group M1 cs:c + 20:.Mgoup) + 151 ozgmup
tree(group) M2 02¢ + 20 WW)
residual M3 0 c

02,0“, = (Ml — M2) / 151 = (14277.00 - 737.79)/151= 39.33
cl... = (M2 —M3) / 2 = (787.79 — 113.60) / 2 = 337.10

 

 

 

 

r2: (:2 ... = 89.33 = 0.1654
2 2 L2 2
o m, + o mg...” + o . 89.33 + 337.10 +113.6O .
h2= 2 02% = 337.10 = 0.7479
cwmmc. 337.10+113.60

28

V. DISCUSSION

The major outcome of this research was the identification of a marker
significantly linked to a putative QTL, termed blm3, controlling bloom time. The late
allele identified (which is contributed by the late blooming parent, Surefire) explained
about 16.5% of the phenotypic variance and delayed bloom time by approximately 15
degree days. Because of the importance of late bloom as a potential escape mechanism
for spring fieeze damage, the goal was to identify late blooming QTL alleles. This allele,
blm3, had the opposite efl’ect of two previously identified bloom time alleles identified in
the sour cherry cross Erdi Boterrno x Schattenmorelle, blml and blm2 (Wang et al.,
2000). The genetic efi‘ect of both blml and blm2 was to promote early bloom time. For
example, blml explained 19.9% of the phenotypic variance and reduced bloom time by
27.8 degree days. Blm2 explained 22.3% of the variation for bloom time and was
detected in two of the three years evaluated (Wang et al., 2000). The presence of early
blooming alleles in the Erdi Boterrno x Schattenmorelle cross was due to the use of the
early blooming Erdi Boterrno as a parent as the early allele, blml, was present in Erdi
Boterrno. In contrast, using a different cross with the late blooming Surefire parent
resulted in the identification of a late blooming QTL.

The lower percent of phenotypic variance exhibited by blm3 as compared to blml
and blm2 could be due to several factors. Because trees are permanent plantings,
replications were accomplished for this study in the form of years — which themselves

were significantly different fi'om each other. Also variation could be explained by lack of

29

tight linkage between E-AAA/M—CGT265 and the blm3 allele. Further replications across

years would reduce this potential for error.

It is not known whether blm3 is found at the same loci as blml, blm2, or the other
bloom time QTL previously identified in Prunus. This is because the linkage map
position for blm3 has not been identified. For example, blml and blm2 were mapped to
Prunus linkage groups EBl and Group2 (Wang et al., 2000), while the major QTL
identified as Lb is located on linkage group 4 (Aruset al., 2003; Ballester, 1998). In an
attempt to target these regions where bloom time QTL had previously been reported in
Prunus, 10, 5, and 3 SSR primer pairs that map to Prunus linkage groups 1, 4, and 2,
were tested (respectively). Primer pairs were selected by proximity to the published QTL
(within 20cM on either side of the estimated QTL). However, none of these SSR primer
pairs identified fragments that were difi‘erentially present between the late and early
bulks. Therefore, AFLP markers were used to try to increase the efficiency of tagging a
QTL location.

AFLP analysis was done using the two standard restriction enzymes, Eco RI and
Mse I, with three selective nucleotides. Subsequently, Eco R1 was changed to use two
selective nucleotides (instead of three), as done in apricot, to identify higher numbers of
polymorphic bands (Vilanova et al., 2003). This change produced comparatively more
bands (as E+AN is 1/4 less selective than the use of E+ANN) although the percent
polymorphic bands was lower. While this proves to be a faster method for screening
primer combinations (16 as opposed to 64), it may not reveal as many polymorphisms
due to co-migration of same sized bands amplified fi'om different regions of the genome.

Given the percent polymorphism evident using three selective nucleotides with the

30

restriction enzyme Eco RI, this population exhibits enough polymorphism to construct a
AFLP linkage map.

Since blm3 is present in Surefire but absent from approximately a third of the
progeny, this marker must be in single dose. However, the lack of fit to a 1:1 ratio
suggests that this marker is on a chromosome region exhibiting extremely skewed
segregation in favor of fragment absence. The skewed ratio is not due to linkage with the
duplicate self-incompatibility loci as a single factor analysis using bloom time and S-
allele marker data was not significant.

Since blm3 explains approximately 16.5% of the phenotypic variance for bloom
time, and confers late bloom, future goals for this project will include the cloning,
sequencing, as well as physical and linkage mapping of this late bloom QTL.
Additionally AFLP analysis could be continued with the goal of identifying additional
markers linked to late bloom QTL.

Identifying markers for bloom time that can be used for marker assisted selection
is not the major long term goal of this study. Although it might be possible to use
markers to eliminate early blooming individuals prior to field planting, and therefore
conserve time and resources, phenotyping for bloom time is very simple and easier than a
genotyping screen. Instead the long term goal is to understand the inheritance and
genetic control of late bloom time in sour cherry. This is because sour cherry exhibits
significantly later bloom time than any other horticulturally important Prunus species,
presumably due to its introgression with the late blooming P. fruticosa. Therefore sour
cherry potentially contains genes/alleles that maximize the bloom delay exhibited in

Prunus. For example, compared to peach, sweet cherry, and apricot, the late blooming

31

sour cherry selections are significantly later blooming. The transgressive segregation
exhibited by the progeny in the Balaton x Surefire cross firrther suggests the loci
controlling bloom time in this allotetraploid species are heterozygous and therefore could
be identified using a QTL approach. Future characterization of the loci that control
bloom time in sour cherry may suggest ways to delay bloom in other early blooming
Rosaceous species, thereby reducing the probability of spring freeze damage and crop

loss.

32

APPENDIX A

Abbreviated SAS input files:

data cherryfinal;

input tree$ year group$ degdays;
cards;

1(66) 4 ' a 209.8
deleted data

4(61) 5 a 266.6

9

run; quit;
proc sort; by group; run;

proc mixed data=cherryfinal;

class group tree;

model degdays = group/outpred=new11 ddfrn=satterth;
random tree(group);

lsmeans group/pdiff adjust=tukey;

repeated /group=group;

run;

proc univariate plot normal;
var resid;
run;

proc plot data=new11;

plot resid*group/VPOS=19 HPOS=50;
plot resid*pred/VPOS=19 HPOS=50;
run;

proc mixed data=cherryfinal method=type3;
class group tree;

model degdays = group tree(group);
lsmeans group/pdifi‘ adjust=tukey;

run;

proc mixed data=cherryfinal;

class group tree year;

model degdays = group year group*year;
random tree(group);

run;

33

APPENDIX B

Figure 1. Frequency distributions of 50% Bloom for the Balaton x Surefire progeny
population over the first 3 years of bloom: 1999 (A), 2000 (B), and 2001 (C). Bloom
date for the parents Balaton (B) and Surefire (S) are shown as inserted letters
correspondingly.

 

1999 Bloom Distribution

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Number of Trees in Bloom
0 .. N or a- m a: ~i on

 

 

318.8 H

 

 

 

 

 

 

 

 

 

 

 

 

 

 

   

NVQI‘NIfiflOQDV’ no
N to O
2§§§a§§§§§§§ r;
DegreeDaysatschloom
A
2000 Bloom Distribution
14 B
512
:10
58
|_
36
34
5
22‘
0..
negro contains-v
3P" ‘0 V
was fiafiifia’a’
Bloom
B

 

 

 

34

B. Figure 1. (continued)

 

 

fibufion

2001 Bloom Di

 

QVNN

«don

J 38

    

 

9".
a:
co
N

 

L . U... . . . .. MAX... . . . (..mvs\...... .xvixiv. It. avast/xix”. V2,... .../”...”;
2...... . » .... .. ... .. . . .. . . ... . .... ... .. I... ..\.. .1 Ti“...
..\H . .\. . . ...-..4‘ &%\\.
......” ... 1 x A. 3...... ........\.\.\.lx\.\.9... k\\.\\“.... w

             
 

        

 

.39 finish... amnion“. .
a.“ .I. / RsvnylflaundelW u” .
mama. .xwuyféfikwwawfl ..

. .H.H.....n.u.u.u.../.

     

 

  

 

 

 

 

 

 

 

 

mmwwmwo

ECO—m c. 089—. no 58532

Degree Days £5096 Bloom

 

 

35

Table 1. Day and degree day comparisons for 50% bloom to identify early and late

APPENDIX C

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

blooming individuals for the early and late bulks (- = no assignment).
1999 2000 2001 2002 2003

letter date hours date hours date hours date hours date hours
a 4/27 187.2 4/24 211.8 4/29 178.7 4/24 179.8 4/28 177 .2
b 4/28 192.4 4/25 216.6 4/30 188.7 4/25 187.7 4/29 186.9
c 4/29 198.8 4/26 221.8 5/ 1 205.3 4/26 189.1 4/30 195.2
d 4/30 205.7 4/27 228.1 - - 4/27 190.8 5/1 202.]
e 5/1 215.2 4/28 234.5 - - 4/28 192.3 5/2 208.1
f 5/2 226. 7 4/29 242.9 - - 4/29 193 .3 5/3 21 1.2
v 5/6 284.5 5/3 276.5 - - 5/8 251 . 1 5/9 253 .7
w 5/7 294.4 5/4 289.7 - - 5/9 257.8 5/10 266.6
x 5/8 304.8 5/5 306.6 5/4 255.9 5/10 263.3 5/11 280.6
y 5/9 310.8 5/6 324.1 5/5 265.3 5/11 268.0 5/12 283.6
2 5/ 10 318.8 5/7 343.4 5/6 274.8 5/ 12 270.7 5/ 13 286.3

 

 

 

 

 

 

 

 

 

 

 

36

 

APPENDIX D

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

£026: 0: 3.0VN N0\5030 9950: o: 5N N
9630: o: ”—030: O: ”530: 0: ON N
F.00N 003030 0.00%IN03030 3N N
P.00N 003030 5.03 NS F030 5.005 503030 VN N
0.v0N 003030 5.03 N9 F030 0. «N F9 ..030 0N N
F.NON 09 F030 5.00— N9 5030 5.02 33030 NN N
Dawn 0000 «N N
0.0¢N 003030 0.53N N03030 0.00N 503030 F.VNO 003030 05 N
N30— 003030 06qu N03030 0.. N
F.00N 003030 5.00.. N9 F030 0. FNN 33030 0. ..NN 003N30 5.. N
N. FNN 003030 500—. N0\ F030 5.02 53030 2 N
5.0mN 003030 5.0 ..N N03030 0 a 33030 mp N
0000 F. 3N N03030 0. wNN 53030 5.00N 003030 9va 003030 3 N
0.v0N 003030 5.0 N N03030 0.30N Fox «030 0 F N
5.3NN 003030 3.0VN N0\5030 0.00N 503030 NP N
0.10N 003030 5.0 N N03030 0.00N 53030 3 N
5.0mN 003030 N.N0IN N03030 0. FNN 33030 or N
0.00N 00\N :30 P. 3N N03030 0.00N «03030 0.000 003030 0 N
5.0mN 003030 3.0VN N0\5030 0. FNN 33030 0.00N 00\ 330 0 N
. 0.00N 003 :30 0.00N N03 :30 0.00N 33030 5.00N 003030 méoN 003030 5 N
N. P N 003030 5.09 N0\ .030 5.00.. 33030 0. FNN 003N30 5.0% 003030 0 N
5.33N 003030 0.00N N03030 5.00? 503030 0. FNN 003N30 N3 N 09 330 3 N
F.00N 003030 0.00N H3030 0.30N F2 F030 v N
5.3NN 003030 0.00N N03030 0.30N 3:030 m .0mN 003030 5 .0NN 003030 00 F
0.00N 003 :30 F. 3N N03030 0.00N 903030 0.000 003030 0.00N 003030 202.6
5.3NN 003030 0.00N N03030 0. PNN _.0\No30 3.03N 003030 0.00N 003030 coaafim
one 5 $03 000 .0 $03 000 5 $00 000 Eco—m DOG Eco—m out. 30¢
000N NO0N ..00N 000N 000 _.

 

“Haggai .30on 800:3 x 53?».— =w .00 CL 8% £80 0202. 0:335 A 935—.

37

D. Table 1. (continued)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

F.00N 09N900 0.00N N03030 0.00N _.9 5030 50 N
5.0NN 003030 0.00N N03030 0. PNN ..9N900 0.00N 090030 0.00N 090030 00 N
0.0 ..N 090900 5.005 N9 5030 0. FN 509N030 5.00N 090030 00 N
5.00N 090030 0.0VN N95030 0.00N 53900 00 N
5.00N 090030 0.0VN N95030 0.00N ..90030 0.00N 009N030 N0 N
N. PNN 090030 0.00N N03030 0. PNN 509N030 ..0 N
0.VON 090030 5.0 ..N N03030 00 N

0000 0000 0000 0000 0v. N
0.0 N 09330 5.005 N9 ..030 0.00N F9 .030 00 N
0.00N 090 :00 0.00N N95030 0.00N ..90030 5v N
v. 3N 095030 5.0 N N03030 0.00N 590030 0 .00N 090030 0* N
5.0NN 003030 5.0 N N03030 0. —N 503030 0.00N 09N900 0.00N 090030 0v N
5.0NN 003030 0.00N N03030 0.00N F9 5030 0.00N 090900 5.0NN 009N030 00 N
v.00N 003030 5.00? N9 _.030 0.00N P9 .030 0.00N 09No30 00 N
0.0 _.N 003030 500—. N9 5030 0.00N 59 P900 0.N¢N 090ng N? N
0.00N 090030 N.NON N03030 p.025: o: 3 N
5.0NN 003030 0.00N N95030 0.00N 590030 00 N
0.00N 09N :00 0.00N N9 _. F30 0.05N ..90030 0.000 095030 00 N
0.00N 090030 0.00N N95030 0.00N 590030 00 N
v.00N 09No30 5.00? N9 ..030 5.00 F 590030 0 .VON 090N900 50 N
v. 3N 095030 0.00N N95030 x20 0.00N 09 F900 00 N
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5.0NN 003030 0.00N N03030 0.00N F9 «030 0 .v0N 090N900 N.0 ..N 09 5030 50 N
0.00N 090030 5.005 N9 ..030 0.00N F9 .030 0.NVN 090N900 00 N
F.00N 09No30 5.005 N9 5030 0 .00N ..9 5030 5.0NN 003030 0N N
5.0NN 003030 N.NON N03030 0N N
000 .0 $00 000 .0 $00 000 a $00 000 5020 000 5020 GB... 301

000N NO0N ..00N 000N 000—.

 

 

 

 

38

D. Table 1. (continued)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

0.00N 003030 N.NON N03030 0. ..NN 33030 0.00N 003030 N 0
N005 003030 0.00 w N03N30 5.00 F 33030 0.0 N 003N30 0.00 w 003N30 0N 0
0.0 N 003030 5.00« N0. 5030 0. PNN 903030 0 w 0
0.0 N 003030 5.00? N0. .030 0.00N ..0. _.030 0.N¢N 003ng 0 P 0
0.00N 003 :00 0.00N N03030 0.00N ..03030 0 .05N 00.0030 5 w 0
N.00F 003030 500— N0. ..030 5.00? 903030 0. FNN 003N30 0.. 0
5.0NN 003030 0.00N N03030 3030.. 0: 0w 0
5.0NN 003030 5.00? N0. 5030 0.030: 0: 3 0
0000 0000 0000 0000 0 .. 0
5.00N 003030 0 .0VN N0\5030 0.00N ..03030 NF 0
0.00N 003030 5.0 ..N N03030 0.00N _.0\0030 0.05N 003030 0 F 0
ONCE ”.030: O: G n
5.00N 00.0030 0.0VN No.5030 0.00N 33030 0 0
0.00N 003030 0.00N N03030 5 0
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UGO—u 2030¢ O... V M
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N00? 0030.3 5.005 N0. F030 5.00.. 503030 F 0
P.00N 003030 N .NON N03030 00 N
F.00N 003030 ...00.. N03N30 00 N
5.0NN 003030 5.0 ..N N03030 0. FNN 33030 #0 N
5.0NN 003030 5.00.. N0. 5030 0. FNN 503030 00 N
0.00N 003030 ... 3N N03030 0. ..NN 503030 0.00N 00. ..030 N0 N
N.00.. 003030 0.05.. N03N30 5.00F 33030 0. ..NN 003N30 N.50.. 00.5300 P0 N
5.0NN 003030 5.009 N0. F030 0.00N P0. P030 00 N
0.0VN 003030 5.0 N N03030 0.00N ..03030 00 N
v. 3N 005030 5.0 N N03030 0.00N 3.0030 0.00N 003030 00 N
000 .0 $00 000 .0 $00 000 .0 $00 GOO 500.0 006 500.0 02.5 30¢
000N NO0N r00N 000N 000 v

 

 

 

 

 

 

 

 

39

D. Table 1. (continued)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

0.00N 003 :00 N.NON N03030 0.00N 3.00.00 0.00N 003030 0.00N 00.10.00 00 0
0.0 3 00.0030 5.00.. N0. ..030 0.00N ..0. 330 0 .v0N 00.0N..‘0 0V 0
0.00N 00.0030 N.NON N03030 0. ..NN ..03030 00 0
5.0NN 003030 5.0 FN N03030 0. ..NN 33030 50 0
0.00 F 00.0N.3 ...005 N0.0N.3 5.005 330:3 0v 0
0.00N 00.0030 5. ..0N N03030 0.00N 3.0030 00 0
0.0.3 00.0030 F. 3N N03030 0.00N 3.0030 5.00N 00.330 0.00N 003030 3 0
5.0NN 003030 0.005 N03030 0. ..NN ..03030 0* 0
5.00N 00.0030 5.0 ..N N03030 0. wNN 33030 0 .00N 0030.3 0 .00N 00.0030 N0 0
0.0 3 00.0030 0.00N N03030 0. PNN 33030 S. 0
5.00N 00.0030 0 .0VN N0.5030 E0300 0: 00 0
0.00N 00.0030 0 .0VN N0.5030 05030.. 0: 00 0
5.00N 00.0030 0 .0VN N0.5030 0.00N 3.0030 5.00N 003030 00 0
...00N 003030 5.0 ..N N03030 0.00N 3. 330 5.00N 00.330 5.0NN 003030 50 0

0.3N 00.0030 5.0 ..N N03030 0. ..NN 33030 00 0
0.0.0 0.0VN N0.5030 0.00N 3.0030 3 0

0.00N 00.0030 0.00N N0.3.00 0.00N 3.330 00 0
5.00N 00.00.00 0.00N N03030 0. FNN 33030 N0 0
5.0NN 003030 0.00N N03030 0. PNN 33030 F0 0
0.00N 00.0030 5.0 3 N03030 0.030.. 0: 00 0
0.0 ..N 00.0030 0.00 F N03030 0.00N 3. 330 ...0NN 00.5N.¢0 N .0 PN 00. 330 0N 0
...NON 00. 330 0.00 w N0.0N.¢0 5.00 .. 3.00.00 0.N¢N 00.0N.¢0 0N 0

v. ..VN 00.5030 0.00N N03030 0. ..NN 33030 0.NvN 00.0N.¢0 5N 0
0.00N 00.0 530 2030.. 0: 0N 0
N55 00.0N.3 0.00 P N0.0N.3 0N 0
N.55 F 00.0N..3 .2005 N0.0N.¢0 5.00 w 330.00 0. .. ..N 00.0N.¢0 v.N0 F 00.0N.¢0 0N 0
0000 0000 0000 0000 0N 0

0.00N 00.0 ..30 N.NON N03030 0.00N 3.0030 0.000 003030 NN 0

COO .0 $00 000 .0 $00 000 .0 $00 000 500.0 COO 500.0 00.... 30¢
000N NO0N ..00N 000N 000 ..

 

 

 

 

 

 

 

 

4o

D. Table 1. (continued)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

0.00N 00.0030 0.00N N0.5030 0 _. 0
0.00N 00.0 _.30 v. 3N N03030 0. FNN 33030 NF 0
5.00N 00.0030 0.00N N0.5030 0. 3N ..03030 0.00N 00. 3.00 .. v 0
p.00N 003030 0.00N N03030 0.00N 3. 330 0r 0
5.00N 00.0030 N.NON N03030 £030.. 0: 0 0
0.00N 00.0030 0.00N N03030 0. 3N 33030 0.00N 003030 5 0
5.00N 00.0030 5.0 _.N N03030 0. 3N 33030 0.00N 003030 0 0
0300 0.030: 0: ”.030: O: m V
0.00N 00.0 _.30 0.00N N0.5030 9030.. 30. 0.05N 00.0030 0 0
5.0NN 003030 0.00N N03030 0. FNN 33030 0 0
0. _.0N 00.5030 5.0 FN N03030 0.00N 3. 330 0.NON 00.0N.00 0.00N 00.0030 N 0
5.0NN 003030 5.0 3 N03030 0. FNN 33030 0.05N 00.0030 w 0
0.00N 003 :00 0.0300 0: £030: 0: 0.000 00.0030 00 0
5.00N 00.0030 0.00N N03030 00 0
0.00N 003030 5.0 3 N03030 0.00N 3.0030 00 0
F.NON 00. 330 0.02 N0.0N.00 0 .00N 3. 330 0.00N 00.00.00 00 0
N02 0030.00 0.00. N0.0N.00 0.00N 3. 330 0.00N 003030 N0 0
0.0 3 00.0030 0.00N N03030 0. FNN 33030 ..0 0
0.030: 305 QGZOC 0: 9.0.50: 0: 00 M
5.0NN 003030 0.00N N03030 0.00N 3. 330 0.00N 003030 0.00N 00.0030 00 0
0.00N 00.0030 0.00N N0.5030 0. EN 33030 00 0
0.0 3 00.0030 5.0 3 N03030 0. FNN 33030 50 0
0.00N 00.0030 500. N0. 330 0.20 00 0
0.00.. 00.0N.00 5.00. N0. 330 0.00N 3. 330 00 0
F.00N 003030 ...00F N0.0N.00 0.00N 3. 330 00 0
0.0 3 00.0030 0.00N N03030 0.030: 0: 00 0
0.00N 00.0030 0.00N N0.5030 0.00N 3.0030 N0 0
0.00N 00.0030 0.00N N0. F F30 0.00N 3.0030 3 0
000 .0 $00 000 5 $00 GOO .0 $00 n50 Eco—m O00 500.0 000.. 30¢
000N NO0N 30N 000N 000w

 

 

41

D. Table 1. (continued)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

5.0NN 003030 0.00N N0.0030 0 .00N .0. .030 0.00N 00.00.00 5.0NN 003030 00 0
N30. 00.00.00 0.00 . N0.00.00 5.00. 3.00.00 0.00N 00. 330 00 0
..00N 003030 0.00N N0.0030 0.00N 3. .030 0.00N 00. .030 N0 0
0.00N 00.0030 5.00. N0. .030 0. .NN .03030 .0 0
5.00N 00.0030 N.NON N0.0030 0. .NN .03030 00 0
0.00N 00.0 .30 N.NON N0.0030 0. ..NN 33030 0 .00N 00.00.00 00 0
0.00N 00.0.30 0.00N N0.5030 0. .NN ..03030 5.00N 00.0030 50 0
0.00N 00.0.30 0.00N N0.5030 0.00N 3.0030 0 .00N 00.0N.00 0.00N 003030 00 0
N.55. 00.0N.00 0.00. N0.0N.00 5.00. 3.00.00 ..0NN 00.5N.00 00 0
0.00N 00.0030 0 .00N N0.5030 0. .NN 33030 ..0N0 00.0030 00 0
0.00N 00.0.30 0.50N N0.0030 0. .NN .03030 N0 0
N00. 00.00.00 ..00. N0.0N.00 5.05. .0.0N.00 0.00N 00.0N.00 .0 0
0.0 .N 00.0030 5.0 .N N03030 0. .NN .03030 00 0
5.0NN 003030 5.00. N0. .030 0.00N .0. .030 0.00N 00.0N.00 0N 0
0.0 .N 00.0030 5.00. N0. .030 0. .NN .03030 ..0NN 00.5N.00 0N 0
0.00N 00.0030 0.00N N0.5030 0. .NN .03030 5N 0
0.00N 00.0030 0.00N N0.00.00 0. .NN 33030 0.00N 00.00.00 0N 0
5.0NN 003030 0.00N N0.0030 0. .NN 33030 0 .00N 00.00.00 0 .00N 00.0030 0N 0
0.00N 00.0 .30 5.05N N03 .30 0.05N .0.0030 ..0N0 00.00.00 0N 0
0.00N 00.0.30 0.50N N0.0030 0.00N .0.0030 0.000 00.5030 0.0.0 00.0 ..30 NN 0
N. . 3 00.0030 0.00 . N0.00.00 0.00N .0. .030 0 .00N 00.0N.00 .N 0
0000 0 .00N N03 .30 0.00N .0.0030 0N 0
5.0NN 003030 0.00N N0.0030 0 .00N 3.330 0. 0
0.0 .N 00.0030 5.00. N0. .030 0.00N 3.330 0. 0
0.030: O: ”.030: O: ”.030: O: Kw Q
0.00N 00.0.30 .. .0N N0.0030 0.00N 3.0030 0. 0
5.0NN 003030 0.00N N0.0030 0.00N 3.330 0 .00N 00.00.00 0 . 0
0.0 .N 00.0030 0.00N N0.00.00 0.00N .0. .030 0.00N 00.00.00 0.00N 00.0030 0. 0
000 .0 $00 006 .0 $00 000 .0 $00 000 500.0 000 500.0 005.. 30¢
000N NO0N 30N 000N . 000 .

 

 

42

D. Table 1. (continued)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

0.0N .. .N 5.0. 5.0N 0.00 >00 .0
_
0.00N 00.0.30 5.05N N03 .30 0.05N .0.0030 0.000 00.5030 0.0.0 00.0.30 .030.
0.503 00.0030 N. .0N N0.5030 0.00N .0.0030 5. .0N 00.0030 0.05N 003030 >00 0?.
0.NON 00.0030 ..0NN N03030 0.0 .N .0. .030 0.NON 00.330 0.00N 00.0030 00E0>0
0.00N 003030 0.00 . N0. .030 .. .0N 3.00.00 0.NON 00.5N.00 0.0 .N 00.00.00 >00 .03
N .55 . 00.0N.00 0.05. N0.0N.00 5.05 . .0.0N.00 0. . .N 00.0N.00 N .50 . 00.5N.00 .00....00
0.00N 00.0.30 0.00N N0.. .30 0.000 0030.00 00 0
0.00N 00.0030 5.0 .N N03030 0.00N .0.0030 00 0
0.00N 00.0030 5.0 .N N03030 0. .NN .03030 0.00N 0030.00 00 0
5.00N 00.0030 0.00N N0.5030 0.030.. 30. 00 . 0
5.00N 00.0030 0.00N N0.5030 0.00N .0.0030 N0 0
0.00N 00.0.30 5.0 .N N03030 0. .NN .03030 0.00N 00. .030 .0 0
0.0.0 0.00N N0.0030 0. .NN .03030 0.00N 00.330 00 0
N00. 00.00.00 5.00. N0. .030 0.030.. 0: 00 0
0.00N 00.. .30 .. .0N N0.0030 0.00N .0.0030 0.000 00.0030 00 0
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0.0 .N 00.0030 5.00. N0. .030 0.00N 3.330 0. .NN 00.0N.00 0.00N 00.0030 00 0
5.0NN 003030 5.0 .N N03030 0. .NN .03030 0.05N 00.0030 00 0
0.0.0 N.NON N0.0030 0.0.0 5.00N 00.0030 0 .00N 00.0030 00 0
0.032. 0: N.NON N0.0030 0.030.. 0.. 00 0
0.00N 00.0030 N .NON N0.0030 0.030.. 0: N0 0
N00. 00.00.00 5.00. N0. .030 0.030.. 0.. .0 0
N00. 00.00.00 0.00N N0.00.00 0.030.. 0: 00 0
..NON 00.330 0.00 . N0.00.00 0.00N 3.330 00 0
N. . .N 00.0030 5.0 .N N03030 0L032. 0: 00 0
5.00N 00.0030 N.NON N0.0030 0. .NN .03030 5.00N 00.0030 0.00N 003030 00 0
0.00N 00.. .30 .. .0N N0.0030 0.00N .0.0030 0.000 0030.00 00 0
000 .0 $00 000 .0 $00 000 .0 $00 000 500.0 000 500.0 00.... 30¢
0 00N NO0N .00N 000N 000 .

 

 

 

 

 

43

APPENDIX E

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

- - - - .8. .8. 2 a: ..8-8-8 8.58..
. .8. .8.

... .8. 8. .8.. 8. .8.. .8. .8..

.8. .8.. .88. .8. .88. .8. .88. .8. - - 8...-8.8 v8.0.0.0
8. 8. 88 8.

88.8.... +8.8... 88.8.... 88.8.... - - 2 8-8-8 88.0.8
.28. .8188. .8188. .8. - - 8...... 8..-8-8 8.8.0....0

8. 8.

8. .8. 8. .8. .8. .8. .8. .8.

88.8. 8...... ......8. 88.8. - - .8182 “...-8.8 880.8
- - - - 2 8 2. ...-8-8 .85....
2 2 2 2 - - 8 83.8.8 895......

8..
.8. .8.
.8.. ....
- - - - 2 .8.. ..o. a. 2-8-8 88.0....

2 .... 2 2 - - 2 83.8.8 888......

v8.8. 8...... 2 v8.8. - - .88... 83.8.8 .85....
8.. .8.
.88. ..8.
.8. .8. .8. .8. .88. .8.
8..... .8.... 8.8. 8. - - 8..... 8.8.8 8.8.0....
8.. .... 8.. .... 8..

8. .8.. .8. .8.. .8. .8.. 8...... 8. .8: .... .8.

.8. .8. .818. .818. .8. .8. .818. .8. .8. 8.. 888-8

.88.... 8.88.... 8.88.... 8.88.8 8.88.... .88.... .... .... 88-8-8 885......
0.... ...... .88.. 08.. 2828 8.8.8 888.. 2:2 0.2:. 89.55....

 

00.80.85 .8000... 0.8 8.5.8.. 05.3.0... 8..... .000 0882 .0.. S... 8:08.... .o 3580... .. 0.0:.

 

E. Table 1. (continued)

 

m... .
.0.». .N0.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

- - - - 8 8 «8-8-8 8.8-8.00
vo...o. 8......
8. ...: .8. .8 .8. .... 2-8-8
- - - - .8. ... .88 .8 ... .8.. .8. .8.-8-8 88-8.00
.... .... .8.
- - - - .8. .8. .8. .8. .818. 2-8-8 8..-.800
8. ..8. 8. ..8.
.88. .8. .88. .8. 8.
- - - - .8. ..8 .8. ..8 .... .8. 88.8.8.8 8.8-89.0:
8. .... .8.-8-8
- - - - 8 .8. .8. 8 88.88 88.88.00
.882.
8.
.8. .88. 8. 8.
.8. .8. .8. .8.: 8. .8. 8. .8. - - .8. .8. 8..-8-8 82..
.8. .8. 8... ...; ...-8-8
- - - - .... .8. .8. .8.. 8 .8.-8-8 .8.)...
- - - - 8.. .88. 8.. 88. 8 88-8-... 8285
8. 8.
88. .8. .8. .8. .88. .8.
88. .8.. .8. .8.. .8.. .8.. .8. .8.. - - 8.. 8-8-8 898......
...-<08.
8. 8.
.8.. .8. 8. .8.. .8.
.8. .8. 8. .8. 8. .8. .8. .8. 8. .8. .8. .8.
.... .8.. .8. .... .8. .... .... .8.. .8. .... .... .8.. .8. .8. 8.-. .-.o 8.68..
.8... ...... .88.. 08.. 2888 :28... .88.. 8a.. 885. ...... be...

 

 

45

E. Table 1. (continued)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

.... ....
.... .8. .... .8. ....

- - - - .8. .8 .8. .8 .... .8. 888-8 .3885
.8. .8. .8. .8. .8. .88.

- - - - .8.... 8..... .81.... ..-8-8 ...-8.00
... 8..

- - - - .8. .8.. ... .... .8. 88.88 8.18.00

8..
8. .8. 8. 8.. .8. .8. .8. 88.8.8
.8. ...: 8. .8. .8. ...: .8. .8. .8. .8. .8. .8. 8. ...: .88-8-8 8..-8.00
.8.... ...... .88.. 08.. 8828 8.8.8.. .23.. 25: 882:. ...... as...

46

APPENDIX F

Table 1. Presence or absence for B-AAA/M-CGT265 fiom all the parents and progeny

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

evaluated.
L Balaton I absent I Surefire I presetfl

1(66) absent 2(55) absent 3(34) present 4(13) absent
2(04) absent 2(56) present 3(36) present 4(14) absent
2(05) absent 2(57) present 3(37) absent 4(15) present
2(06) absent 2(58) present 3(38) present 4(16) ‘ present
2(07) present 2(59) absent 3(3 9) absent 4(18) absent
2(09) present 2(60) absent 3(40) absent 4(19) absent
2(10) absent 2(61) absent 3(41) Jresent 4(21) absent

2(1 1) absent 2(62) absent 3(42) absent 4(22) present
2(12) present 2(63) absent 3(43) absent 4(24) present
203) absent 2(64) absent 3(45) present 4(25) absent
2(15) absent 2(65) absent 3(46) absent 4(26) absent
2(16) absent 2(66) absent 3(47) present 4(27) present
2(17) absent 3(01) absent 3(48) absent 4(28) present
2(18) present 3(02) absent 3(49) absent 4(29) absent
2(19) present 3(03) absent 3(50) absent 4(30) absent
2(22) absent 3(05) present 3(51) present 4(3 1) absent
2(24) absent 3(07) present 3(52) absent 4(33) Jresent
2(28) absent 3(08) present 3(53) absent 4(34) absent
2(29) absent 3(10) absent 3(54) absent 4(35) absent
2(30) absent 3(12) absent 3(55) absent 4(37) absent

2(3 1) absent 3(14) absent 3(57) absent 4(40) absent
2(32) present 3(15) present 3(58) present 4(41) absent
2(35) present 3(16) absent 3(59) present 4(42) absent
2(36) present 3(17) absent 3(60) absent 4(43) absent
2(37) present 3(19) absent 3(61) absent 4(44) absent
2(38) absent 3(20) present 3(62) absent 4(45) absent
2(40) absent 3(21) present 3(63) absent 4(46) absent
2(41) absent 3(22) absent 3(64) absent 4(49) present
2(42) absent 3(24) absent 3(65) present 4(50) absent
2(43) absent 3(25) absent 4(01) absent 4(51) absent
2(45) absent 3(26) present 4(02) absent 4(52) absent
2(46) present 3(27) present 4(03) absent 4(54) present
2(47) present 3(28) present 4(04) absent 4(55) absent
2(48) absent 3(29) absent 4(07) absent 4(56) absent
2(50) absent 3(30) absent 4(09) absent 4(58) present
2(51) absent 3( 3 1) absent 4(10) absent 4(59) absent
2(52) present 3(32) absent 4(11) present 4(61) absent
2(53) absent 3(33) absent 4(12) absent

Balaton = absent Surefire = present Progeny = 106 absent and 45 present (Total = 151)

47

 

APPENDIX G

Table 1. SAS output for “group” comparison where group a is absent for E-AAA/M-

CGT265 and b is present (therefore representing blm3 and BIm3 respectively, includes

 

 

 

 

2002 and 2003).
Least Sguares Means
Effect Group Estimate Std Error DF t Value Pr >j t 1
group a 220.08 1.9483 141 112.96 < .0001
group b 235.21 3.0052 137 78.27 ' < .0001

 

 

 

 

 

 

 

 

48

 

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A“

“j“,