Investigating the dwarfing phenomenon in apple and the
identification of genetic markers for a novel source of
powdery mildew resistance
Audrey DIDIER
DESS management of biodiversity:
Methodology of study and genetics resources valorisation
2001/2002
Summary
The major characteristics of commercial apple varieties are durable resistance
resulting in a reduction in pesticide application, a high yield combined with a low harvest
cost and high fruit quality. To achieve these desired traits, molecular markers can be
employed as useful tools in the pyramiding of resistance genes or in the marker assisted
selection of genes of interest. Powdery mildew is one of the most important diseases affecting
apple production worldwide. This study used bulked segregant analysis with RAPD markers
to identify a genetic marker linked to a new source of powdery mildew resistance in Aotea. In
addition the same technique was used to position a locus thought to be involved in the control
of the dwarfing phenomenon in the commonly used dwarfing rootstock M9. The genetic
markers identified in this study will be used to further characterise powdery mildew
resistance and dwarfing in apple. This information will be valuable to future marker assisted
selection in apple.
Resumé
Les propriétés majeures des variétés commerciales de pomme sont considerées
comme étant une résistance durable entraînant la diminution des applications de pesticide, un
haut rendement associé a un faible coût de récolte et une haute qualité de fruit. Pour acquérire
ces charactéristiques, les marqueurs moléculaires peuvent être employés comme des outils
utiles dans le pyramidage de genes de résistance ou dans la sélection assistée par marqueur
pour les genes d’interêt. L’oïdium est une des plus importantes maladies affectant la
production mondiale de pomme. Cette étude a utilisé la bulked segregant analysis avec des
marqueurs RAPD pour identifier des marqueurs génétiques liés à de nouvelles sources de
résistance à l’oïdium chez Aotea. De plus, la même technique a été utilisée pour placer un
locus que l’on croit être impliqué dans le contrôle du nannisme chez une des rootstocks
naines les plus communement utilisée M9. Les marqueurs génétiques identifiés dans cette
étude pourront être utilisés pour une caractérisation plus approfondie de la résistance à
l’oïdium et du nannisme chez la pomme. Ces informations pourront être précieuses pour de
future sélection assistée par marqueurs chez la pomme.
Key words: Powdery mildew; dwarfing; resistance gene; molecular markers; BSA; RAPD
2
TABLE OF CONTENTS
INTRODUCTION.................................................................................................................... 4
1 - Powdery Mildew............................................................................................................... 6
2 - Dwarfing ........................................................................................................................... 7
Planting system in apple production.................................................................................. 7
Used of dwarfing rootstocks in apple ................................................................................ 8
Knowledge on genetic control of the dwarfing ................................................................ 10
3 - Introducing the research for this project ......................................................................... 12
MATERIALS AND METHODS .......................................................................................... 13
1 - Varieties used and crosses .............................................................................................. 13
2 - Populations ..................................................................................................................... 14
Powdery Mildew (PM) ..................................................................................................... 14
Dwarfing (DW) ................................................................................................................ 15
3 - DNA extraction............................................................................................................... 16
4 - DNA quantitation ........................................................................................................... 17
5 - Pre-screening markers .................................................................................................... 17
6 - Bulked Segregant Analysis (BSA) ................................................................................. 18
7 – Mapping ......................................................................................................................... 19
RESULTS ............................................................................................................................... 20
1 - Powdery Mildew............................................................................................................. 20
Pre-screen markers .......................................................................................................... 20
Bulked Segregant Analysis ............................................................................................... 20
Mapping ........................................................................................................................... 21
2 - Dwarfing ......................................................................................................................... 21
Bulked Segregant Analysis ............................................................................................... 21
Mapping ........................................................................................................................... 23
DISCUSSION ......................................................................................................................... 24
ACKNOWLEDGEMENTS .................................................................................................. 30
REFERENCES ....................................................................................................................... 31
APPENDIX ............................................................................................................................. 35
3
INTRODUCTION
The main objective of breeding programmes in apple is to combine high fruit quality,
high yield and resistance to known major pests and disease. This objective originates from
the demands of producers who require durable resistance and a reduction in costs associated
with chemical treatment and also consumer demand for a lower degree of pesticide
application (Gardiner et al., 2002). Traditionally, crosses are carried out between commercial
varieties and wild species known to possess major resistance genes (Janse et al., 1994).
Crosses are successful if the character is sexually transmissible, under the control of major
genes and simply inherited (Dayton, 1977). However, several backcross generations are
necessary before the resistance can be integrated into a desirable cultivar of high fruit quality.
For a species like apple which has a lengthy generation time (5 years), long juvenility period,
is self-incompatible, this method is long and complex (Lawson et al., 1995). In addition,
using this traditional method breeders cannot select plants which express two or more
resistance genes because of the epistatic interactions between the genes (Gardiner, 2002).
However, this is often a desirable breeding objective as the presence of more than a single
resistance gene against the same disease or pest in the same variety could reduce the ability of
the pest or disease to overcome the resistance. There are two types of resistance to pests and
diseases: resistance controlled by major genes (vertical) and resistance controlled by many
resistances genes (horizontal). In the case of vertical resistance, the level of inheritance is
high, around 50 %, but it is easier to breakdown by mutation within the pest or pathogens
(Bus et al., 1998). Polygenic resistance is more stable, but the resistance is highly variable in
the progeny and weakly heritable (5-15 %).
Breeders require tools to understand the inheritance of resistance genes to enable them
to select plants efficiently and at an early stage of development (Bus et al., 2000). Marker
assisted selection using markers which are close to and flank the gene of interest has been
used successfully in several crops (Gardiner, 2002). This method halves the time taken to
introduce new genetic characters into high quality varieties particularly when the trait of
interest is usually determined phenotypically at a late developmental stage (cf. figure 1), or
when pyramiding several genes (Kellerhals et al., 2000). The use of marker assisted selection
in a breeding program also reduces orchard maintenance costs as those plants not carrying the
genes of interest can be eliminated at the seedling stage.
4
The development of new cultivars possessing several resistances to the same or
different diseases will result in a decrease in fungicides and pesticides used by the producer
(Gomez-Lim, 2002). This will in turn reduce the cost of such chemical treatments, the
quantity of fungicides and pesticides in the soil and in the environment, and slow the
development of fungal resistance to pesticides, thus answering current consumer demands.
Fischer (2000) presented an interesting example of two cultivars ‘Rebella’ and ‘Regine’,
which were bred at the institute for fruit breeding at Dresden-Pillnitz. These two dessert
apples possessed a combination of resistance genes to major pests and disease like apple
scab, and fireblight, including powdery mildew for ‘Rebella’. These resistances enabled an
80 % or more decrease in fungicide used for both varieties. In addition to these new
resistances, these varieties are also good pollen parents for other commercial varieties, which
could be a useful tool for resistance gene pyramiding.
As well as their use in marker assisted breeding programmes for the
development of new resistant cultivars, markers linked to resistance genes can also be used to
begin fine mapping and eventual cloning of the resistance genes. Once cloned the resistance
genes can then be introduced into the cultivar of interest by transformation. However, actual
results show that successful transformation in apple is difficult and the expression of the
resistance is highly variable in the progeny obtained (Hanke et al., 2000).
Identification of resistance source
Inheritance studies
Genetic markers
Markers assisted
selection
Traditional
way
Cloning
New cultivar
Fig. 1: Marker assisted selection
5
1 - Powdery Mildew
Powdery mildew (Podosphaera leucotricha (Ell. & Ev.) Salm.) is one of the most
important diseases infecting apple resulting in significant economic loss by decreasing yield
and fruit quality. This fungus, which is an obligate biotrophic can infect buds, blossoms,
leaves, twigs and fruit (Markussen et al., 1995) with different consequences. The infection
begins on the lower surface of leaves with the appearance of whitish felt-like patches
(Ndabambi et al., 2000). When the fungus spreads the leaves become narrow, crinkled,
stunted and brittle, twigs stop growing and become stunted, blossoms abort and contaminated
buds produce the infected leaves and blossoms next year. Fruits develop russetting when the
infection attains a high level. Damage caused by the disease is also dependent upon climate
of the growing area (Markussen et al., 1995).
Some genetic studies have been carried out on the molecular and phytopathological
characterisation of this fungus (Lespinasse et al., 2000) as part of the Durable Apple
Resistance in Europe (D.A.R.E.) project. The author showed that several physiological races
of P. leucotricha exist in different European countries, but characterisation of the races is
difficult as the fungus is an obligate biotroph. In spite of the development of an invitro
method for the inoculum conservation (Lespinasse et al., 2000), results of glasshouse
inoculation do not always correlate with observed field resistance (Kellerhals et al., 2000).
Most of the time, the plants are classified into different phenotypic classes after natural
infection, which is in turn dependent upon meteorological conditions (Janse et al., 1994).
Markussen et al. (1995) demonstrated that the phenotype obtained in the first year (in field or
glasshouse) is not always in agreement with the phenotype obtained in the field on older
material and therefore the development of molecular markers will be important for the
accurate selection of early resistant individuals. So far, six resistance genes have been
identified (Alston et al., 2000) of which, two originate from wild species of apple: Pl1 in
Malus robusta and Pl2 in Malus zumi (Markussen et al., 1995). The principal SCAR markers
known for Pl1, which have been widely used in the past for the breeding are OPAT20450 and
OPD21000. These flanking markers at 4.5 cM and 5 cM respectively, obtained by bulked
segregant analysis using RAPDs have been used in marker-aided selection (Markussen et al.,
1995). For Pl2, two SCAR markers: OPN18 and OPUO2, have been determined by the same
method, but they are further away at 7cM and 8 cM respectively (Bus et al., 2000). In the
D.A.R.E. project two other resistance genes: Plw and Pl-D12 have been mapped by the same
6
technique. Eight SCAR markers for Plw and seven for Pl-D12 have been found (Lespinasse et
al., 2000).
Until now, the major sources of resistance to powdery mildew came from wild Malus
species. To identify different resistances to powdery mildew, a German fruit genebank at
Dresden-Pillnitz has been created with wild species and hybrids and maintained without
fungicide spray since 1981 (Buttner et al., 2000).
2 - Dwarfing
Planting system in apple production
Some centuries ago, an ideal apple tree was defined as having the lower branch high
enough that a rider could pass underneath, i.e. between eight and ten metres high
(Muggleston, 1994). But with the development of apple production and its growth as a
worldwide crop, this definition is no longer true. Indeed global apple production has
increased 54% in 30 years (1970-2000) and 31% in 10 years (1990-2000) in the world (FAO
source). For such production, a high tree is not ideal in the field because of the manual
harvest cost. This production increase was linked with a higher density of trees in the field.
However, with the increase in the number of trees grown per hectare, producers established
that fruit maturity became delayed and the development of size and colour of the fruit
decreased (Widmer et al., 2001). The reduction in fruit size is negatively correlated with the
number of trees per hectare, the number of fruit per tree and tree volume. This variation of
fruit size due to a high planting density can be accepted for some apple cultivars which
produce big fruit but not for those which have medium fruit like ‘Royal Gala’ (Weber, 2001).
The number of trees per hectare equally has an impact on the growth and the yield. For
example, doubling the number of trees in one area can result in a 45% decrease in trunk
cross-sectional, resulting in inadequate root growth and too smaller trees (Widmer et al.,
2001). For the harvest, the yield per tree is reduced but it increases per hectare. The problem
is that this increase is not proportional to the number of trees in the field. So a high density
system is not ideal for long term production because it does not offset the financial
investment resulting from a larger number of trees. Taking into account the “optimal
combination of natural, technical and financial resources”, Weber (2001) defined an optimal
7
planting density of between 3000 and 4000 trees per hectare dependent upon soil conditions
and variety grown.
Used of dwarfing rootstocks in apple
To grow apple trees at a higher density and so reduce vegetative growth which is
expensive to remove and can decrease the fruit quality and the yield, a chemical technique
has been used (daminozide) (Samad et al., 1999). This treatment results in a reduction of
vegetative growth, a higher fruit colour and stronger fruit buds. However, consumer
preference dictates a reduction in pesticides, and a demand for more “natural” fruit. Over
time breeders have observed and identified dwarfed apple trees in their natural environment
which were studied for a long time, to know find how such trees could be avoided. Because
in their natural state such trees are inappropriate for commercial use, the best way to
introduce this character is to use this tree like a rootstock. The choice of the rootstock in
apple breeding is very important because it can determine growing spread, tolerance at
different soil and environment, resistance to soil disease, insects or pest, scion compatibility,
fruit quality (texture, organic acid, sugar concentration, etc,) and yield. In addition, root
function is also modified by soil type (O2, CO2 concentration, humidity, temperature, etc.)
and the variety grafted to the rootstock (Westwood, 1993). Varieties grafted onto dwarfing
rootstocks develop less vegetative growth without reduction to the yield. However, the
absolute size of the mature tree graft onto the dwarf rootstock will change with soil, climate,
orchard density and scion variety, which corresponds to the specific balance of the rootstockscion system (Westwood, 1993). An ideal dwarf tree habit is a height of 1.7 metres, with 8 or
10 feathers and between 0.80 and 1.10 metres above the ground (Coleman, 2000). This
process of grafting rootstocks is now commonly used in apple production and many
rootstocks are available to the breeder that are dwarfing (cf. table 1).
One of the most commonly used rootstocks is Malling 9 (M9 see chapter Materials
and Methods) which was described for the first time in a paper in 1917 (Tukey, 1964). This
rootstock has improved the fruit quality, precocity and yield of the scion associate (Fischer,
2001). A very interesting study (Samad et al., 1999) compared the vegetative growth, the
number of buds, flower and fruit, with the use of different interstock bridge grafts (M9
dwarfing rootstock, same cultivar or with nothing). They obtained in the second season, a
higher floral density, percentage of blossom buds, number of fruits and blossoms by bud,
8
yield and a decrease of the twig length with the M9 bridge grafting than with others. The
grafting treatment decreases vegetative growth and increases reproductive growth, which is
amplified when the M9 rootstock is used.
Nevertheless M9 has some limitations, primarily its susceptibility to the major apple
pests and disease like fireblight, woolly apple aphid and brittle wood (Crassweller et al.,
2001). Other dwarfing rootstocks have been developed and compared to M9. 5 rootstocks
bred at the Dresden-Pillnitz institute (Fischer, 2001), which are a Pillnitzer supporter 1’®;
Pillnitzer supporter 2’®; Pillnitzer supporter 3’®; Pi-Au 51-11 and Pi-Au 56-83, showed
excellent fruit quality, early production, a better yield with a lower crown volume than M9,
and scrab resistance. However, no fireblight resistance was observed and only medium
resistance to mildew was noted. The major problem of these new rootstocks is that this result
varies with the environment the tree is grown in and none of these rootstocks perform as
consistently as M9.
Table 1: Comparisons of apple rootstock characteristics from Pennsylvania State college of agricultural sciences
web site
Rootstock
Size class
Fire blight
Collar rot
VD
VS
MS
Budagovsky 146
VD
S
R
Malling 27
VD
S
R
Poland 22
VD
VS
MR
Budagovsky 491
Poland 16
VD
S
MR
Poland 2
D
S
R
D
S
R
Budagovsky 9
D
R
R
Geneva 16
D
R
R
Geneva 65
D
VS
R
Malling 9*
D
S
MR
Mark
D
S
R
Ottawa 3
SD
R
Unknown
Vineland 1
SD
VS
S
Malling 26
SD
R
R
Geneva 11
SD
R
R
Geneva 30
SD
MR
MR
Malling 7
SV
S
VS
Malling Merton 106
SV
R
R
Malling Merton 111
SV
R
R
Malling 2
V
Unknown
Unknown
Seedling
V
Unknown
Unknown
Budagovsky 490
*Refers to NAKB 337 clone of M9
Size class: VD= very dwarf; D= dwarf; SD= semi-dwarf; SV= semi-vigorous; V= vigorous
Resistance class: VS= very susceptible; MS= moodily susceptible; S= susceptible; R= resistant; MR= moodily
resistant; VR= very resistant
9
Knowledge on genetic control of the dwarfing
Little is know about the genetic control of dwarfing, and the mechanisms involved in
controlling the dwarfing effect. To date, no genetic markers for genes controlling the
dwarfing phenomenon have been identified in apple. Understanding the number of genes
involved in controlling this trait and identifying markers for them could have a significant
impact on the breeding of apple rootstocks.
Alston (1976) carried out the most important study concerning the genetic
determination of dwarfing. He defined three types of dwarf and analysed the segregation of
each type. He concluded that several genes determined the dwarfing ability of the rootstocks.
-
Early dwarf:
Dwarfing appears four weeks after the germination and growth until 120mm
in the first season. Plants had little internode and did not survive if the winter was too
rough. In twenty-one progenies, he observed three different segregation: 3:1 (vigorous
: dwarf); 7:1 and 15:1. Two independent recessive genes could explain this
segregation and he gave the hypothetical genotype of each parents implicated in
crosses (cf. table 2).
Table 2: Parental genotype assumed by observed phenotype segregation
Segregation
3:1
7:1
7:1
Parent’s name
642
OR33T90
Howgate Wonder
3151
A46/28
Parent’s genotype
assumed
*D1d1 d3d3
or
d1d1 D3d3
641
649
1002
1381
D1d1 D3d3
D1d1 d3d3
or
d1d1 D3d3
15:1
A1584
James Grieve
Starkrimson
Starking
Golden delicious
Starkspur Golden Delicious
Cox
TSR1T187
D1d1 D3d3
* D= dominant gene; d= recessive gene
However, the presence of two recessive genes did not explain the spur-type
habit which seemed to be genetically independent from the early dwarf genes. The
spur-type habit seemed to be linked to another recessive gene d4 which is capable of
10
replacing either d1 or d3. Therefore, the mechanism induced dwarf tree with spur-habit
could be controlling by three genes with at least two at the recessive state.
The progenies of one cross ‘James Grieve’ x ‘TSR1T187’ which
corresponding in a segregation 3 vigorous : 1 dwarf, showed a regrowth proprieties
when they were planting in field, up to two-thirds the height of normal seedlings in
50% of the plants. A similar result was obtained with the cross ‘Golden Delicious’ x
‘0-521’. Like ‘0-521’ and ‘TSR1R187’ were related, the author concluded in the
possible presence of a common dominant gene for regrowth promotion carried on this
both varieties.
-
Crinkle dwarf:
This plant could grow between 300 to 600 mm in two years. They had normal
internodes and small rounded crinkled leaves. This kind of dwarf tree was observed
only in two progenies of the cross ‘Irish Peach’ x ‘TSR1T187’. The segregation on
the progenies was 3:1 (vigorous : crinkle dwarf) which corresponding to a single
recessive gene when the both parents used for the cross are vigorous.
-
Sturdy dwarf:
This kind of dwarf could growth until one metre in three years, seldom more.
The characteristics of this plant is a long juvenile period, a high number of branching,
small internodes and could be selected eight weeks after the germination just before
placing in field. This phenotype seemed to be control by several recessive genes at at
least two loci, which could be carried by Malus robusta seedling MAL 59/1,
‘Jonathan’ and ‘Cox’.
The effect of the genes was apparent at different time: the d1, d3 and d4 after 4 weeks
of germination, the sturdy dwarf genes after 8 weeks, the regrowth gene in the field at about
12 weeks and the crinkle dwarf gene in the second growing season. Therefore, several genes
specific to particular development stages could be involved in growth in apple.
11
3 - Introducing the research for this project
The main objective of this study was to find molecular markers for the two traits of
interest described previously: resistance to powdery mildew and the dwarfing ability of
rootstocks. This research was carried out using Bulked Segregant Analysis (BSA)
(Michelmore et al., 1991), where pools of individuals derived from a single population
segregating for the character of interest, are compared using molecular markers. This method
has already been used for gene identification and mapping and notably in apple breeding
research (Bus et al., 2000; Gardiner et al., 1997; Markussen et al., 1995). Apple is highly
heterozygous due its self-incompatible nature and so characters almost always segregate in
initial crosses between two polymorphic cultivars. That is why the individuals used for this
analysis were the result of a cross between two individuals and not F2, or backcross progenies
like in the other biological models. Markers identified in this way using the powdery mildew
and dwarfing population could, after conversion to SCAR markers become useful tools in
marker assisted breeding programmes. As the work carried out on the genetic control of
dwarfing is an initial investigation in this area, the results obtained will be useful in
understanding the number of genes involved and generate markers to test the results in larger
populations.
Fig. 2: Comparison between a dwarf tree (right) and a vigorous tree (left) in the third growing year
12
MATERIALS AND METHODS
1 - Varieties used and crosses
Aotea
This rootstock was developed and released from the HortResearch apple
rootstock breeding programme. It was selected for a good disease resistance, low fertility and
clay soil (Muggleston, 1994). This rootstock is compatible with all the commercial apple
cultivars, has a sound and rapid root development, earlier production, tolerance to
Phytophthorum cactorum and Peniophora sacrata (root canker), reduced incidence mildew,
scale and blackspot and is resistant to woolly apple aphid.
Malling 9
This rootstock which was known since 1879 like Yellow Metz (Turkey, 1964),
was bred at the East Malling Research Station in U.K. It is commonly used in Europe, and
produces 2 to 3 metres high trees (Muggleston, 1994). The characteristics are an early bearing
the first or second year planted, an early cropping, amelioration of fruit quality, resistance to
apple scab and mildew, but susceptible to fire blight and woolly apple aphid (Bus, 1994).
However, M9 needs a heat treatment before be used like a rootstock which is
available and known as (FKV) M9 (Muggleston, 1994).
Robusta 5
Robusta 5 was breed in the Arnold Arboretum in U.S and produces vigorous
tree, tolerant of winter cold, shows good resistance to mildew and apple scab, and strong
resistance to fire blight and woolly aphid (Bus, 1994).
Cross Aotea x M9
This population has already been used to find markers linked to the woolly
apple aphid (WAA) resistant gene, Er3. SCAR markers were obtained and known as
OPA011250
bp,
OPE011350
bp,
OPO051700
bp
which are at 3.3 cM, 7.6 cM and 0.8 cM
respectively (Gardiner et al., 1997).
13
Cross M9 x Robusta 5
Rootstocks were generated from the M9 and Robusta 5 cross, and the
Braeburn scions were grafted onto these rootstocks for phenotypic assessments. Leaves were
harvested from cuttings taken from corresponding M9 and Robusta 5 individuals kept in the
nursery.
2 - Populations
Trees were grown in Hawkes Bay on the East Coast of the North Island in New Zealand.
Leaves were taken from each individual and stored at -70 °C.
Powdery Mildew (PM)
This population was derived from a cross between Aotea, the resistant parent and
M9 the susceptible parent. The population consisted of 283 individuals, with 151 resistant
plants (0), 92 susceptible (1-5) and 43 dead (X) without determinate phenotype. The
phenotype of the plants is observed in the field, where the plants are submitted to the natural
pressure of PM. Therefore some plants classified as resistant could be susceptible plants that
have escaped infection.
160
140
120
100
80
60
40
20
0
X
0
0+
1s
2s
3s
4s
5s
Fig.3: Segregation of the different phenotypes in the Aotea x M9 population
Susceptible individuals could be divided into five classes ranging from 1 to 5 (cf. figure
3), depending on the type and extent of reaction to PM. If the plants showed a strong
susceptible reaction, they are classed as highly susceptible; class 5.
14
The segregation of resistant (0) to susceptible (1-5) was significantly different from a 1:1
ratio (2obs = 13.90; p> 0.001). We would expect to observe a 1:1 ratio if the resistance to PM
was determined by a single major gene. Therefore the segregation within the Aotea x M9
population indicated that the control of this resistance is due to several genes. Comparing the
segregation to that expected within the population if the resistance was controlled by two
genes (3:1) or three genes (5:3) showed that the segregation was significantly different from a
ratio of 3:1 (2obs = 21.87; p> 0.001) but not significantly different from the ratio of 5:3 (2obs
= 0.03; p> 0.50). Therefore three genes seem to be implicated in the control of the resistance
to PM. However if the dead plants are considered as susceptible plants, we obtained a chi2
value that was not significantly different to a 1:1 ratio (2obs = 0.79; p> 0.30). The study of
this population could give a better idea of the genetic control involved in this resistance.
Dwarfing (DW)
This population is a cross between M9, conventially used as a dwarfing rootstock
and Robusta 5 a non-dwarfing parent. This population is small, with only 101 individuals
segregating for 16 dwarf, 9 semi-dwarf, 17 intermediate and 59 vigorous. It is good to note
that this is the first year of data for this population. The main objective of this study was to
indicate the number of genes involve in controlling DW. However as the population is small,
the results will have to be confirmed by the screening of more trees.
If we look at the segregation, considering that dwarf and semi-dwarf belong to the
same class and intermediate and vigorous in a second class (cf. figure 4), we obtain a 1:3
ratio (2obs = 0.01; p> 0.90) of dwarfing plus semi-dwarfing to intermediate plus vigorous.
This ratio indicates that two genes could be involved in the genetic control of dwarfing.
We obtained the same result when only the results for dwarfing and the vigorous
individuals were used in the test (2obs = 0.06; p> 0.50).
15
Vigorous +
Intermediate
Dwarf +
Semi-Dwarf
Vigorous
Intermediate
Semi-dwarf
Dwarf
90
80
70
60
50
40
30
20
10
0
Fig. 4: Segregation between Dwarf and Vigorous in Robusta 5 x M9 population
3 - DNA extraction
We extracted 96 samples of the population Aotea x M9. The remaining individuals of
this population had been already extracted.
The leaves were kept frozen with liquid nitrogen, in a plastic bag. After the leaves
were ground, 2 ml of extraction buffer (cf. Appendix 1) was added to the bag. The contents of
the bag was mixed and put in an eppendorf with 400 l of chloroform:octanol (24:1 v/v).
Samples were mixed and incubated at 65° C for 30 minutes, timed from the last tube going
in. After incubation the samples were mixed and placed on ice for 10 minutes then spun in a
Sorval at 4 °C for 10 minutes (12 K). The top layer was removed, placed in a fresh tube, and
1.0 ml of ice cold isopropanol was added to each tube. The mix was left on ice for 30 minutes
or more and spun in an eppendorf centrifuge for 5 minutes at full speed. After the supernatant
was tipped off, the pellet was washed with ice cold 70% ETOH (1.0 ml) and left overnight in
the fridge. Then ethanol was discarded and the DNA was dried in speed vacuum for
approximately 3 minutes. The size of each pellet was evaluated by eye and if the pellet was
small, 50 l of distilled water was put in a tube, 60 l for a medium, 75 l for the large one.
The pellet was resuspended overnight in the fridge.
16
4 - DNA quantitation
Two methods were used for the quantitation depending upon the sample number.
When the number was less than 30 samples, a quantitation was carried out by hand. Initially a
dilution of 1:5 (2 l DNA to 8 l distilled water) was made. Two further dilutions were made
from this 1:5 dilution, one at 1:2 and one at 1:5 with SBx10 (Sample Buffer; cf. Appendix 1)
and distilled water. After incubating for 10 minutes at 65° C and spinning briefly, the
dilutions and marker lanes were loaded in a 0.9% agarose gel in 1xTAE buffer with ethidium
bromide (EtBr) (1.53g agarose in 170 ml buffer; cf. Appendix 1) and run for 1 hour at 60
Volt. The concentration was determined by comparison between the DNA and a lambda
phage DNA standard (concentration: 50 g/l).
When the number was more than 30 samples, we used the Biomek 2000 (robot). An
original dilution of 1:10 (2 l DNA to 18 l distilled water) was made. Two dilutions were
made by the robot from this 1:10 dilution, one at 1:2 and one at 1:5. The plate was heated for
10 minutes at 65 C. 20 l of samples and 5 l of marker lanes, were loaded into agarose gels
in 1 x TAE buffer (2.4 g agarose in 270 ml TAE buffer) at 70 V. for 1 hour. After migration,
gels stained in EtBr for one hour. Again the concentration of DNA was estimated by
comparing DNA dilution with the lambda standard on the gel.
A 1 g/l dilution of each sample was made.
5 - Pre-screening markers
Only the population derived from the cross between Aotea x M9 was pre-screened
because there are at present no genes identifed for dwarfing that could be screened for the M9
x Robusta 5 population. 4 SCAR markers for known powdery mildew resistance genes were
tested (cf. Table 3). 76 individuals from the Aotea x M9 population were selected on the basis
of phenotype and DNA concentration (for example, individual with a DNA concentration
equal to 20 g/l were not selected), both parents and two controls, one positive and one
negative were also included. Products of PCR (cf. Appendix 2) were loaded onto agarose gels
17
in 1 x TAE buffer at 70 V. for 1.30 hour. After migration, gels were stained in EtBr for one
hour. The correlation between marker and phenotype was then analysed.
Tab. 3: SCAR markers used for the pre-screening of Aotea x M9 population.
Size
PM gene
Parent
Marker
Pl1
Robusta 5
AT20 F/R
450
60
Pl2
A689
U2 F/R
1500
65-55
Plmis
Mis o.p.
AC20 F/R
1700
70-60
Pl2
A689
F2P2N F/R
400
52
bp
Ta
Notes
Also detects Plmis at 2000bp
6 - Bulked Segregant Analysis (BSA)
For each bulk, DNA from 12 individuals was mixed together (100 l of each dilution
1 g/l). Samples for each bulk were selected according to phenotype (cf. Appendix 3 & 4).
For each population, 4 bulks were constructed: two of one extreme and two of an opposite
extreme e.g. resistant versus susceptible, dwarfing versus vigorous. 520 RAPD primers,
which are 10-base random sequences (Michelmore et al., 1991), were tested. 48 primers were
set up at one time in a v-bottom PCR plate. 3 l of primer was added to 22 l of water, 15 l
of master mix (cf. Appendix 5) and 1.5 l of bulks DNA. After add 18 l of paraffin oil, the
plate was covered and placed in a PCR machine (cf. Appendix 2). PCR products were loaded
onto two component gels (1.2 g agarose, 1.2 g amplisize in 270 ml TAE buffer) for two
hours, put in EtBr for one hour and rinsed in distilled water for 1 hour.
Fig.5: Example BSA results
18
From the results, primers were identified that amplified a band in both resistant
bulks but not in the susceptible bulks or vice versa (cf. figure 5). After identifying potential
primers linked to the genes by BSA, each interesting primer (12 per run), was tested on both
parents and 30 individuals which composed the bulks (cf. figure 6). The presence or absence
of the band of interest was scored with a plus or minus, respectively and the percentage of
recombination was calculated. If the score was lower than or equal to 36 % of recombination,
the primer was tested on all the individuals of the population available. 4 primers could be
tested at the same time on 96 individual in a v-bottom PCR plate.
Fig.6: Results with the 30 individual screen
7 – Mapping
All the primers used on all the populations were tested to show if they followed
Mendelian segregation with a 2 test. If such segregation was not found, we deduced the
primer was probably affected by segregation distortion.
The program Join Map v. 3.0 (Van Ooijen et al., 2001) was used to determine genetic
linkage. Two maps were constructed for each population studied; for example an M9 map
was generated from loci that were heterozygous in the M9 parent and similarly a Robusta 5
map was created from loci that were heterozygous in the Robusta 5 parent. It will be possible
to integrate the two maps after further studies using co-dominant markers. The log of odds
ratio (LOD score) determined the statistical significance of the results and the probability that
the loci were genuinely linked rather than linked by chance. Loci linked at LOD 2 were
considered not strongly linked. The Kosambi mapping function was used to determine
genetic distance (Kosambi, 1944).
19
RESULTS
1 - Powdery Mildew
Pre-screen markers
No segregation was found in the Aotea x M9 population using primers AT 20
F/R, AC 20 F/R and F2P2N F/R. For the primer U2 F/R, half the resistant individuals
possessed a band at 2000 bp and half did not (cf. table 4) but most of the susceptible
individuals were missing the same band. Only four individuals possessed the band out of 38
samples.
Table 4: Segregation obtained by the U2 F/R pre-screening in function of the phenotype
Resistant
Susceptible
+
17
4
21
-
21
34
55
38
38
76
Bulked Segregant Analysis
Of the 509 primers tested on the PM bulks, 38 showed some interesting results (cf.
Appendix 6). After screening the primers on the 30 individuals, 9 primers appeared linked to
the PM gene with a percentage of recombination lower than 35 %. For the majority, the band
was present in the resistant individuals (8 of 9). The screening of all the population showed
that only one primer AE10 seemed to be linked to the resistance gene. The 2000 bp band
amplified by AE 10 seemed to be linked to the gene controlling PM resistance (cf. table 5).
Table 5: Percentage of recombination of the primer OPAE 10 in Aotea x M9 population
Band
Screen on the 30 individuals
Screen on the all population
500
20 %
48.20 %
1050
30 %
45.29 %
2000
36.67 %
24.89 %
In order to have an idea about the number of genes involve in the genetic control
of the resistance, AE 10 was also tested on the dead plants (cf. table 6). The band was present
20
in one third of the individuals. This result showed that such dead plants could not be placed
with the susceptible plants and so the hypothesis of three genes involved seems to be more
likely.
Table 6: Absence/ presence of the 2000 bp band in the Aotea x M9 population in function of the phenotype.
0
0+
1
2
3
4
5
X
+
44*
2
11
9
16
27
7
12
128
-
102
3
3
1
3
2
4
22
140
146
5
14
10
19
29
11
34
268
*Number of individual
Mapping
The results of linkage analysis on loci heterozygous in Aotea showed that 2
pairs of loci were linked, one at LOD 10 and the other at LOD 3. The other loci were
unlinked. However, none of these loci were linked to the resistance gene. JoinMap linked the
locus AE 102000 that was tested on more individuals, to the resistance gene at LOD 10. The
estimated distance between this locus and the gene was 24.9 cM.
None of the loci heterozygous in M9 were linked to the resistance gene.
2 - Dwarfing
Bulked Segregant Analysis
As the number of dwarfing individuals was low some individuals were included in
a single bulk more than once (cf. Appendix 7). The BSA showed 75 primers were interesting.
Of these 75 primers, 27 were tested on all the population available (cf. table 7). 2 primers,
RAPD 30 and RAPD 35, were not tested because the new stock received, did not work. A
DNA ladder 100 bp was used for determinate the interesting band’s position.
For this population, some primers were tested on all the population whenever the
recombination percentage was high because no markers were already identified for dwarfing
and because the segregation of the band was interesting. For some primers, the band seemed
to be strongly linked with the dwarf individual (e.g. 4 plus and 11 minus) but not with the
vigorous (e.g. 7 plus and 8 minus), and vice versa (cf. table 8).
21
Table 7: List of primers used on all the population
Primer
Band
Screen on
Screen on
Size
30 individuals
all the population
(bp)
RAPD 2
RAPD 3
RAPD 6
RAPD 7
RAPD 8
RAPD 9
RAPD 10
RAPD 13
RAPD 14
RAPD 16
RAPD 19
RAPD 21
RAPD 22
RAPD 26
RAPD 29
RAPD 30
RAPD 31
Primer
27.27
27.27
31.82
30
18.18
31.03
36.36
34.48
36.36
34.48
30
23.33
36.36
30
40
30
36.67
33.33
33.33
36.67
46.67
31.51
37.84
44
36.23
33.8
41.89
36.48
37.33
44.59
31.95
26.67
33.33
32
41.89
28
Not tested
37.33
37.33
21.33
Screen on
Size
30 individuals
RAPD 35
RAPD 39
RAPD 41
RAPD 46
RAPD 48
RAPD 50
RAPD 54
RAPD 55
RAPD 56
RAPD 68
RAPD 69
RAPD 75
700
300
700
700
400
700
900
1100
450
700
1500
1100
600
1500
1500
500
600
1000
900
Screen on
all the
population
% Recombination
(bp)
% Recombination
700
400
1300
2000
700
1500
1200
2000
700
1200
500
1200
1000
300
900
1300
800
700
800
1500
Band
20
40
40
48.27
48.28
41.38
34.48
24.14
33.33
26.67
36.67
30
36.67
27.27
30
26.67
30
36.36
Not tested
47.3
45.95
45.33
44
46.66
45.33
26.67
37.33
40
46.66
42.67
49.33
33.8
20
34.66
37.5
30.66
Table 8: Example of primer with a high percentage of recombination; RAPD 39 (700bp)
Dwarfing
Vigorous
+
4
7
11
-
11
8
19
15
15
30
These kinds of primers were tested on more individuals to see if this segregation
stayed the same when all the individuals were screened. 6 primers: RAPD 6, 10, 16, 26, 41700
bp & 900 bp,
54 and 551500 bp, upon the screening of more individuals, showed that these primers
did not amplify bands linked to dwarfing. However, 8 other primers: RAPD 7, 14, 22, 39 300bp
& 700 bp,
41400 bp & 1100 bp, 50 and 55600 bp, kept this pattern of segregation after the screening. We
found the same kind of segregation in the primers RAPD 2, 8 and 48, which had initially a
low percentage of recombination. Primers where the presence of a band was linked to
dwarfing and the absence to vigorous and vice versa were used in linkage analysis.
22
Mapping
For the Robusta 5 data, two main groups, and three pairs of linked loci were
obtained with JoinMap, with the other loci unlinked. In one of the main groups, the eleven
loci remained together up to LOD 3. When the LOD score was increased one locus was
eliminated from the linkage group, and the others stayed together until LOD 9. In this group a
high number of loci showed a segregation distortion (8 of 11). In the other main group, five
loci were linked together up to LOD 2. When the LOD score was increased, the group of loci
broke up into unlinked loci. The mapping of the main group was done by eye, because the
software was not able to do this, probably because of segregation distortion observed in these
loci. The dwarfing locus could not be positioned on this main linkage group, shown in figure
7A.
RAPD 211000
RAPD 91500
1.9
RAPD 751100
12.4
14.9
DW1
RAPD 291300
10.3
RAPD 31700
2.9
7.3
RAPD 191200
7.9
RAPD 46450
3.9
RAPD 19500
7.9
RAPD 68600
RAPD 691000
A
B
Fig. 7: Genetic maps found with JoinMap software, estimated distances were expressed in cM.
A: Robusta 5 map; B: M9 map
For the M9 map, two groups of 4 and 2 loci respectively were obtained with
the JoinMap software. Loci within these two groups were linked at LOD 10. The other loci
were found to be unlinked. We obtained the map showed in the figure 7B. Two loci flanked
the dwarf gene at 14.9 cM and 7.9 cM respectively.
23
DISCUSSION
Powdery Mildew
The pre-screening of Aotea x M9 population showed that Pl1 and Pl2 did not
seem to be present in this population. For the resistance gene Plmis, the primer AC 20 was not
linked to the resistance phenotype, which suggested the probable absence of this gene.
However, with the primer U2, the band (2000 bp) explained susceptible the individuals but
only half of the resistant. This result could mean that the gene Plmis was present in this
population. For verifying the presence of this gene, the screening of more individuals will be
required. However, the absence of link between the SCAR markers and the phenotypes of
individuals tested cannot allow as to conclude that the corresponding genes were not present
in the populations studied. Indeed, each SCAR markers was developed for a particular
population, and so, the gene could be in the population without the associated marker.
The result with the primer AE10 on all the population including the dead
plants showed that the hypothesis of three genes was more likely to explain the resistance to
powdery mildew in this population. In this case, individuals required a dominant allele at one
locus (i.e. A) to be resistant. If individuals were homozygous for recessive alleles at this locus
then dominant alleles were required at two additional loci (B and C) for expression of
resistance to powdery mildew. This could happen if, in the absence of A, B codes for a
product, which is necessary in the resistance process but alone is not enough to result in
resistance, therefore requiring the additional product of C.
The mapping showed that the primer AE102000 bp was strongly linked to the
resistance gene because it stayed linked at LOD 10 and was 24.9 cM from the gene of
interest. The AE102000 bp locus, although not close to the resistance gene will be helpful for
future studies, to place new loci. Furthermore, this genetic marker is the first one identified
for powdery mildew resistance in Aotea.
Plants were classified into different phenotypic classes after observation of the
plants in field which had been submitted to the natural disease pressure of the fungus. This
casts doubt on the real phenotype of the plant. Indeed, a plant could be classified as resistant
class but actually be susceptible because it had escaped infection. This uncertainty could be a
24
problem in the BSA technique, because we were not totally sure of the phenotype of the
individuals used to construct the bulks. If the number of misclassified individuals is low, the
effect on the BSA result is minimal and markers could be found, but if many individuals were
misclassified could mean that the bulks were not representative of the true resistant and
susceptible individuals. An answer to this problem will be the development of an inoculation
method, but this will be hard to realise because powdery mildew is an obligate biotrophic and
because results in the glasshouse do not always correlate with observed field resistance
(Kellerhals et al., 2000). The evaluation of the phenotype over several years could be a way
to limit the number of misclassified individuals. However, the level of infection depends of
the meteorological conditions and the fungus race, which could vary each year. Dayton
(1997) reported the existence of a highly resistant cultivar which showed susceptibility in
meteorological conditions highly favourable to the pathogen. A middle phenotype could
therefore be more appropriate.
Dwarfing
The analysis of the phenotypic segregation in dwarfing population M 9 x
Robusta 5 showed that two genes could be involved in this mechanism. After simplification
of the number of classes (dwarfing + semi-dwarfing versus intermediate + vigorous), three
hypothetical model could be proposed to explain this segregation (cf. figure 8).
Model 1: This model was based on the existence of two genes with recessive
alleles in the dwarfing parent and a vigorous parent, which was heterozygous for the two
genes. The individuals in the progenies were vigorous if they had at least one dominant
vigorous allele. This model is in accord with the work of Alston (1976) on the segregation of
early dwarf.
25
Model 1
Dwarfing (DW)
Vigorous (V)
M9
x
Robusta 5
aa bb
Aa Bb
Aa Bb : Aa bb : aa Bb : aa bb
V
DW
DW expressed when homozygous for recessive alleles at DW locus
Model 2
Dwarfing (DW)
Vigorous (V)
M9
x
Robusta 5
Aa bb
aa Bb
Aa Bb : Aa bb : aa Bb : aa bb
V
DW
V
V
V expressed when dominant allele B, overcomes A, the DW allele.
Model 3
Dwarfing (DW)
M9
Vigorous (V)
x
Robusta 5
Aa Bb
aa bb
Aa Bb : Aa bb : aa Bb : aa bb
DW
V
DW expressed when dominant allele at both loci A and B
Fig. 8: Hypothetical segregation model for explain the 3 : 1 ratio in the population M 9 x Robusta 5
26
Model 2: In this model, dwarfing phenotype was determined by a dominant
allele (i.e. A) which is present at the heterozygous state in the dwarfing parent and another
gene with two recessive alleles. The vigorous parent did not present the dwarf allele and
therefore had recessive alleles for this gene, but had a dominant allele (i.e. B) that
determinate the vigorous character at heterozygous state. In the progenies, individuals were
vigorous if they had the recessive alleles for the two genes, the dominant allele B and the
recessive allele for the gene A, or when the dominant allele B overcame the dwarfing
dominant allele A. The presence of the dominant dwarf allele and the absence of the
vigourous allele B resulted in the dwarf phenotype.
Model 3: The last model explained the dwarf phenotype by the presence of two
dominant alleles at two loci (i.e. A and B) in the dwarfing parent at heterozygous state. For
obtain the dwarf phenotype in the progenies the both dominant alleles was required. The
individuals were vigorous when they had one of the dominant alleles or the both recessive
alleles.
The plants from the cross M9 x Robusta 5 were phenotyped this year for the first
time. Therefore, the classification of each plant was linked to the environmental conditions in
the field (soil, climate, etc.) and the growth rate could be variable according to the individual.
The segregation of the marker loci showed that the semi-dwarf individuals could be grouped
with the dwarfing in a same phenotypic class. However, the intermediate individuals seemed
to be a mix between the dwarfing and the vigorous plants.
The DW1 locus on the M9 map shown in figure 7B, was placed initially on basis of
the dwarfing and semi-dwarfing individuals. The locus remained in the same position and
approximate distance when the vigorous individuals were introduced. However, some
vigorous individuals (10) had the M9 alleles and were therefore expected to belong to the
dwarf class. These individuals are probably vigorous due to a second gene involved in this
mechanism and could be used in further studies to identify markers for the predicted second
gene. In addition, the semi-dwarf individuals showed the M9 genotype at marker loci
surrounding DW1 and could be grouped in the dwarf class, the intermediate individuals were
a mix between M9 or Robusta 5 genotype. This result confirmed the previous hypothesis of
two phenotypic classes.
27
The dwarfing gene could not be positioned on the Robusta 5 map, because the linkage
was not clear. More data is necessary to place DW1 on this map. From the linkage data of the
Robusta 5 map, we might predict its position to be above the RAPD 291300.
The M9 x Robusta 5 population was also small with few dwarf individuals and
therefore, results obtained by this study will have to be confirm on a larger sample. The
phenotypic classification needs to be repeated over several years to confirmed the hypothesis
of these two classes and permit the accurate classification of intermediate individuals in one
of two classes. Tree phenotypes were determined this year at the end of their second growing
season in the orchard so the trees were 3 years old. It is possible that the trees will change
phenotype as they become older and accumulate different chemical processes. Larger visual
differences between trees will facilitate the classification of the intermediate individuals and
make it easier to classify individual as dwarf or vigorous.
The study carried out by Alston (1976) involved a higher number of genes than our
model, and their effect occurred at different stages of plant development. Our models of two
genes could be simplistic, but we need to understand what happens at the physiological level
to have a better understanding of the dwarfing mechanism and see more clearly the genetic
process that could involved in the dwarfing phenomenon. The genetic markers identified in
M9 as linked to the dwarf phenotype in this study are the first markers to be identified as
associated with dwarfing. These results will form the basis of a broad investigation into the
dwarf phenomenon.
The use of the BSA method associated with the RAPD technique has allowed the
testing of a high number of primers (520) in a limited time. This method can be applied on
individuals coming from one cross segregating for the gene of interest. In addition, as apple is
highly heterozygous, it is possible to carry out this type of analysis on a F1 population and
this is a great advantage for plant with long generation time. However, this method is based
on the plants phenotype, and therefore the success of this technique is dependent upon the
confidence we can place on the different phenotypic classes. For example, with the powdery
mildew resistance, we were not sure that the resistant plants were really resistant or if they
had just escaped to the infection.
The RAPD technique is fast and does not need a high quantity of DNA and is cost
effective. A disadvantage of this method is its reproducibility, i.e. the ability to repeat the
28
results obtained with this technique between the different laboratories. However, this can be
overcome by converting the close primers linked to the gene of interest to SCAR markers,
which are less sensitive to variations in amplification conditions due to longer sequences.
Such markers will be tested on all the populations to confirm the results obtained.
The work carried out on powdery mildew resistance as part of this study is the
beginning of a long process which could result in markers for breeders to use in pyramiding
genes of interest. However, even if polygenic resistance is more difficult to break down, good
management of this resistance be required to guarantee a durable resistance. Several authors
(Fischer, 2000; Jones, 2001) advise cultivating mixed plants in a single area which have
different susceptibility to the pathogen (i.e. resistant and tolerant plants) to preserve the
stability of the host-pathogen system. The existence of a population heterozygous for
resistance genes but which has the same commercial quality could decrease the epidemic rate,
the susceptibility at virulent race of pathogen and the fitness of the parasite. Conversely, if
the pathogen infects a plant which has a specific gene of resistance, it will evolve until it
acquires the corresponding avirulence gene. However, such a mixed population is hard to
obtain with apple tree due to the use of clones in production and in addition known
resistances have been already exploited for several commercial cultivars. Breeders now need
new types of resistance and this is one of the main interests of the conservatory like the Fruit
Genebank at Dresden-Pillnitz in Germany, which is used for research into powdery mildew
resistance. In this institute, they evaluate the resistance proprieties and the fruit quality of
wild species and hybrids maintained without fungicide spray since 1981 (Fischer, 2000). This
condition allows the conservation of the genetic diversity of different varieties of apple tree
and help to identify new resistance genes. The use of fungicides does not seem to be totally
avoided. In fact, two or three applications of pesticides when the infection is greater could
avoid significant infection and the resultant breakdown of the resistance (Fischer, 2000). The
gene bank could be a valuable reservoir of varieties that may be of interest as the market
evolves and breeders needs change.
The research of molecular markers linked to resistance genes, is part of a more global
effort, which includes the characterisation of the resistance status of a large number of apple
varieties, analysis of the variability of the pathogens, determination of the genetic
mechanisms involved in resistance, the development of new breeding strategies and the way
these new resistant cultivars are perceived by the consumer.
29
ACKNOWLEDGEMENTS
I would like to thank all the staff of HortResearch who was very friendly with me and
helped me to fell like at home.
The Dr Sue Gardiner, who accepted me in her laboratory and gave me the opportunity
to lived in New Zealand for few month.
Jo and Heather for them friendship and the many laugh we had together, and Mike for
his apparition time to time, it was a pleasure to work with you.
A very special thanks to Rachel, who supported me during the manipulation and the
report redaction. I very enjoyed working with you, thanks for your smile, your friendship,
your explication on English expression (or vocabulary), and your availability with all the
questions I had... I was expected a supervisor, I had found more than that, I found a friend. I
will miss you….
I would thanks my family for their support, my friends from the DESS, thanks for
your nice e-mail, I hope that we will keep in touch, and Raphael for your visit surprise, your
friendship and the moments “stay Zen” in the south.
And the last but not the least, is for you Marion, thanks for all the happy moments we
had, your friendship and your support during all the stage. It was a pleasure to live and visit
the country with you. I cannot summarise in few words all that I would express to you and
what you represent now for me… So thanks…
Audrey
30
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33
Internet sites
FAO
http://apps.fao.org/cgi-bin/nph-db.pl?subset=agriculture
Pennsylvania State college of agricultural sciences
http://tfpg.cas.psu.edu/tables/table1-7.htm
34
APPENDIX
Appendix 1
Extraction buffer (for 1 L)
Sorbitol 2M
Tris pH 7.5 1M
70 mls
220 mls
EDTA 0.5M
NaCl 5M
44 mls
160 mls
cTAB
8g
(hexadecyl trimethylammonium bromide; break down of cells)
n-lauroyl sarcosine
distilled water
10 g
(sodium salt)
500 mls
TAE buffer
With ethidium bromide
TAE
40 mls
(242 g Tris, 57.1 ml Aceolic acid, 100 mls EDTA)
ethidium bromide (0.5 mg/ml)
distilled water
3 mls
1957 mls
Without ethidium bromide
TAE
40 mls
(242 g Tris, 57.1 ml Aceolic acid, 100 mls EDTA)
distilled water
1960 mls
Sample buffer 10 x Concentration (3 mls)
EDTA 0.5M
1.25 ml
SDS 10 % (warm to dissolve)
80 l
Ficoll
1.5 g
Bromophenol Blue 0.4 %
315 l
(add KOH to dissolve)
distilled water
1.75 ml
35
Appendix 2
PCR programme
Pre-screenning
With one Ta
With a touch-down
Stage 01 (1 cycle)
Stage 01 (1 cycle)
T = 94° C
T = 94° C
Time = 2'45''
Time = 2'45''
Stage 02 (40 cycles)
Stage 02 (20 cycles)
T = 94° C
Time = 0'55''
T = 94° C
Time = 0'55''
T = Ta
Time = 0'55''
T = Ta 1
Time = 0'55''
T = 72° C
Time = 1'39''
T = 72° C
Time = 1'39''
Stage 03 (1 cycle)
Stage 03 (20 cycles)
T = 72° C
T = 94° C
Time = 0'55''
T = Ta 2
Time = 0'55''
T = 72° C
Time = 1'39''
Time = 10'00''
Stage 04 (1 cycle)
T = 72° C
Time = 10'00''
RAPD
Stage 01 (1 cycle)
T = 94° C
Time = 2'45''
Stage 02 (40 cycles)
T = 94° C
Time = 0'55''
T = 37
Time = 0'55''
T = 72° C
Time = 1'39''
Stage 03 (1 cycle)
T = 72° C
Time = 10'00''
36
Appendix 3
Bulks composition
Phenotype
AK 840
0
AK 845
DNA concentration
DNA concentration
Individual
Phenotype
250
AK 813
5
150
0
100
AK 841
5
100
AK 878
0
250
AK 876
5
40
AK 879
0
100
AK 887
4
250
AK 881
0
200
AK 889
5
40
AK 882
0
100
AK 894
5
80
AK 883
0
175
AK 916
5
60
AK 888
0
100
AK 918
5
100
AK 898
0
200
AK 920
4
250
AK 906
0
250
AK 947
5
150
AK 923
0
200
AN 8
5
250
AK 926
0
175
AN 16
5
70
AK 861
0
90
AK 846
4
80
AK 862
0
90
AK 897
4
80
AK 880
0
90
AK 899
4
40
AK 904
0
100
AK 903
4
100
AK 905
0
100
AK 915
4
100
AK 914
0
100
AK 930
4
60
AK 917
0
100
AK 932
4
70
AK 922
0
100
AK 946
4
60
AK 945
0
100
AK 998
4
80
AK 948
0
90
AN 2
4
80
AK 952
0
90
AN 3
4
200
AK 955
0
90
AN 31
4
70
ng/ul
Suceptible Bulk 1
Individual
Susceptible Bulk 2
Resistant Bulk 2
Resistant Bulk 1
Aotea x M9
ng/ul
37
Appendix 4
Bulks composition
M9 x Robusta 5
DNA concentration
Individual
ng /ul
DNA concentration
ng /ul
100
AJ 56
110
AJ 59
90
AJ 60
100
AJ 74
80
AJ 71
80
AJ 81
100
AJ 79
60
AJ 86
90
AJ 99
60
AJ 93
100
AJ 108
100
AJ 113
100
AJ 116
50
AJ 118
70
AJ 138
80
AJ 147
100
AJ 177
110
AJ 158
50
AJ 195
40
AJ 178
100
AJ 46
100
AJ 196
110
AJ 50
100
AJ 76
120
AJ 71
80
AJ 83
90
AJ 82
100
AJ 100
70
AJ 84
70
AJ 109
80
AJ 115
100
AJ 121
80
AJ 116
50
AJ 135
40
AJ 119
60
AJ 141
80
AJ 134
120
AJ 156
80
AJ 177
110
AJ 168
100
AJ 183
70
AJ 190
100
AJ 191
90
Dwarfing Bulk 1
AJ 41
Dwarfing Bulk 2
Vigorous Bulk 2
Vigorous Bulk 1
Individual
38
Appendix 5
Master mix for 384 BSA set up (for 48 primers)
Distilled water
5157 l
Formamide 1%
51.6 l
10 x PCR buffer
706 l
dNTPs
353 l
Magnesium
TAQ polymerase (50 units)
173.05 l
40 l
39
Appendix 6
Primers used during the experimentation on Aotea x M9 population
Interesting
Primers with
BSA
OPAA 9
OPAA 10
OPAB 3
OPAB 5
OPAD 4
OPAD 19
OPAE 10
OPAG 7
OPAI 13
OPAI 17
OPAJ 3
OPAL 1
OPAL 13
OPAL 20
OPAP 7
OPAP 19
OPAQ 4
OPAQ 20
OPAQ 19
OPAR 2
OPAR 4
OPAR 5
OPAR 9
OPAR 15
OPAR 17
OPAR 17
OPAR 18
OPAR 19
OPAR 20
OPAN 20
OPAS 14
OPAS 19
OPAT 8
OPAT 13
OPAU 1
OPAU 2
OPAV 2
OPAW 15
OPAX 2
Band presents in
Vigorous
Dwarfing
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
Band size
bp
1400
500/1200
1500
1200
1100
600
500/1050/2000
1500/2000
300
1500
1000
900
500
400
1500
2000
900
500/600
500
1300/1500
900/1300
500/1000
400/800
800
1300
800/1000
400
1100
1300
400/800
2000
600
1000
1750/2000
900
1500
1100
1000
600/1100
Interesting primers
with screen on 30
individuals
Band size
bp
X
X
X
900
600/1000
500/900/1050/2000
X
300/400/900/1000
X
X
1000/1300/1500
1000
X
X
700/1200/1300
400/800
X
1400
X
400/700
Note: The size of the band of interest in some cases varied between the BSA and the screen
on the 30 individuals. Also some bands, which were not observed with BSA were noted in
the screen 32 as having a pattern of interest and were therefore scored.
40
Appendix 7
Primers used during the experimentation on M9 x Dwarfing population
Interesting primers
with BSA
Band presents in
Vigorous
RAPD 1
Band size
Dwarfing
Bp
X
400/800
Interesting primers
with screen on 30
individuals
Band size
Bp
RAPD 2
X
1500/1750
X
700
RAPD 3
X
400/800/1500/1750
X
400
RAPD 4
X
400/1500
RAPD 4
X
1300
RAPD 5
X
2000
RAPD 6
X
1200
X
1300
RAPD 7
X
600
X
2000
RAPD 8
X
800
X
700
RAPD 9
X
800
X
1500
2000
X
1200
RAPD 10
RAPD 11
X
X
500/900/1400
RAPD 12
X
2000
RAPD 13
X
1500/2000
X
2000
500/1300
X
800
X
1200
X
500/1200
RAPD 14
X
RAPD 14
X
1500
RAPD 15
X
500
RAPD 16
X
1400
RAPD 17
X
1300
RAPD 18
X
1400
RAPD 19
X
500/1100
RAPD 20
X
1500
RAPD 21
X
1100
X
1000
RAPD 22
X
400/1200
X
300
X
900
RAPD 23
X
1500/2000
RAPD 24
X
1000/1500/2000
RAPD 25
X
500
RAPD 26
X
600/1000
RAPD 27
X
500
RAPD 28
X
1500
RAPD 29
X
900
X
1300
RAPD 30
X
600
X
800
41
Appendix 7 (Next)
Primers used during the experimentation on M9 x Dwarfing population (Next)
Interesting primers
Band presents in
Band size
with BSA
Vigorous
RAPD 31
X
1000/1500/1750
RAPD 32
X
1100/1200/1500
RAPD 33
Dwarfing
X
bp
X
700
X
300/700
300/1500
X
400/900/1100
900
X
700
X
450
X
700
X
1500
1500
X
1100
RAPD 35
X
300
RAPD 36
X
600
RAPD 37
X
400
900
RAPD 38
X
500
RAPD 39
X
800
RAPD 41
X
X
RAPD 41
X
1500
RAPD 42
X
400/600/800
RAPD 43
X
600
RAPD 44
RAPD 44
X
X
RAPD 45
2000
700
X
1400
RAPD 45
X
700
RAPD 46
X
1100
RAPD 47
X
1300
RAPD 48
X
500
RAPD 49
X
400/500/600
RAPD 50
X
900/1000/1300
RAPD 51
X
800
RAPD 52
X
600/1000
RAPD 53
X
500
RAPD 54
Bp
1300
500
RAPD 40
individuals
Band size
700/800/1500
X
X
with screen on 30
X
RAPD 34
RAPD 38
Interesting primers
X
RAPD 55
X
1500
X
600/1500
RAPD 56
X
1500
X
1500
RAPD 57
X
400
42
Appendix 7 (Next)
Primers used during the experimentation on M9 x Dwarfing population (Next)
Interesting primers
with BSA
Band presents in
Vigorous
RAPD 58
Band size
Dwarfing
bp
X
1500
RAPD 59
X
600
RAPD 60
X
800
RAPD 61
X
700/900
RAPD 62
X
600/1200
RAPD 63
X
400
RAPD 64
X
1100
RAPD 65
X
700
RAPD 66
X
600
RAPD 67
X
400
RAPD 68
X
500
RAPD 69
X
Interesting primers
with screen on 30
individual
Band size
bp
X
500/600
X
1000
X
900
1750/2000
RAPD 69
X
1100
RAPD 70
X
500/600
RAPD 71
X
2000
RAPD 72
X
1500
RAPD 73
X
1300
RAPD 74
X
800
RAPD 75
X
1100
43