MARKER ASSISTED BREEDING AND SCREENING OF

APPLE SCAB RESISTANCE (VF GENE) FROM

COLUMNAR APPLE SEEDLINGS BY PCR

Anel Bekbergen

MSc Thesis

Green Biotechnology and Food Security

University of Eastern Finland

Faculty of Science and Forestry

Department of Environmental and Biological Sciences

September 23, 2016

UNIVERSITY OF EASTERN FINLAND

Faculty of Science and Forestry, Department of Environmental and Biological Sciences

Green Biotechnology and Food Security program

Anel Bekbergen: Marker assisted breeding and screening of apple scab resistance (Vf gene)

from columnar apple seedlings by PCR

MSc thesis, 48 pages

Supervisors: Harri Kokko, MSc and Anna Toljamo, MSc

September 23, 2016

________________________________________________________________________

Key words: apple scab, Venturia inaequalis, Vf gene, columnar apple, DNA markers

ABSTRACT

Disease-free and commercially profitable apple production requires adequate cultivation and

breeding processes. Apple breeding is typically a lengthy and cost consuming process. Apple

cultivation is affected by the climate change and results in the introduction of pathogens and

the existence of apple scab. Apple scab is an economically significant disease in apple-

growing regions with high precipitation in spring and summer time that damages fruits and

leaves. If uncontrolled, apple scab may result in the reduction of fruit quality and quantity and

in severe infections, almost complete devastation of apple crop.

To prevent the apple scab, fungicide treatments are a requisite in commercial orchards.

However, due to high costs and environmental risks, fungicides are not an optimal control

method. A more effective and environmentally friendly approach to cope with apple disease is

using resistant cultivars. The Vf resistance gene, derived from wild species Malus floribunda

821, has been the generally used gene in apple breeding programs against apple scab. Markers

associated with resistance genes can be used to significantly fasten the selection process of

resistant cultivars.

The aim of this study was to detect the Vf gene from columnar apple seedlings by using PCR.

DNA extraction from the fresh leaves of apple seedlings was performed by CTAB method.

PCR reaction was carried out using Vf primers followed by the electrophoresis to detect the

samples with Vf genes. The Vf gene was detected from four apple seedlings. In three out of

these four samples, it was observed that Vf genes were combined with the columnar growth

type. Results of this study indicate that PCR reaction process is a useful way to detect the

apple scab resistant Vf genes.

УНИВЕРСИТЕТ ВОСТОЧНОЙ ФИНЛЯНДИИ

Факультет естественных наук и лесного хозяйства, Кафедра окружающей среды и

биологических наук

Программа Зеленая Биотехнология и Пищевая Безопасность

Анель Бекберген: Маркер-ассоциированная селекция и скрининг резистентности (Vf

ген) к парше столбчатых саженцев яблони посредством ПЦР

Магистерская диссертация, 48 страниц

Руководители: Харри Кокко, MSc и Анна Толиамо, MSc

Сентябрь 23, 2016

________________________________________________________________________

Ключевые слова: парша яблони, Venturia inaequalis, ген Vf, столбчатые яблони, ДНК

маркеры

АБСТРАКТ

Парша яблони, вызванная аскомицетом Venturia inaequalis, заражает плоды и листья и

является широко распространенным заболеванием в яблочно-развивающихся регионах.

Если своевременно не контролировать, парша яблони может привести к снижению

качества и количества фруктов, а при тяжелых инфекциях почти полного опустошения

урожая яблок.

В промышленных садах для предотвращения парши яблони за один сезон требуется до

пятнадцати противогрибкового лечения. Тем не менее, из-за высоких затрат и

экологических рисков, фунгициды не являются оптимальным решением в борьбе с

заболеванием. Использование устойчивых сортов обличается более эффективным и

экологически чистым подходом, чтобы справиться с болезнью яблока. Vf ген

устойчивости, выявленный от диких видов т.е. Malus floribunda 821, был наиболее

широко используемым геном в селекционных программах яблони против парши. Для

значительного ускорения процесса отбора устойчивых к болезни сортов, могут быть

использованы молекулярные маркеры связанные с генами устойчивости.

Целью данного исследования было обнаружить Vf ген из столбчатых саженцев яблонь с

использованием ПЦР. Столбчатые яблони имеют толстый стебель, короткие ветви и

междоузлия, что позволяет насаждать с высокой плотностью с меньшим количеством

обрезки в садах. Выделение ДНК из свежих листьев саженцев яблони проводили

методом CTAB. Реакцию ПЦР проводили с использованием Vf праймеров с

последующим электрофорезом для обнаружения образцов с Vf геном.

Vf ген был обнаружен в четырех саженцах яблони. В трех из этих четырех образцов,

было обнаружено, что Vf гены были объединены со столбчатым типом роста.

Результаты этого исследования показывают, что ПЦР является эффективным способом

обнаружения Vf генов резистентности к парше.

ШЫҒЫС ФИНЛЯНДИЯ УНИВЕРСИТЕТІ

Жаратылыстану ғылымдары және орман шаруашылығы факультеті, Қоршаған орта

және биология ғылымдары кафедрасы

Жасыл Биотехнология және Тағам Қауіпсіздігі бағдарламасы

Анель Бекберген: ПТР арқылы бағаналы алма көшеттерінен алма таз қотыры ауруына

төзімділікті (Vf ген) тексеру және маркер көмегімен іріктеу

Магистрлік диссертация, 48 бет

Ғылыми жетекшілері: Харри Кокко, MSc және Анна Толиамо, MSc

Қыркүйек 23, 2016

________________________________________________________________________

Түйін сөздер: алма таз қотыры, Venturia inaequalis, Vf гені, бағаналы алма көшеттері,

ДНҚ маркерлері

ТҮЙІНДЕМЕ

Алма таз қотыры (қоздырғышы Venturia inaequalis) алма шаруашылығы дамыған

аудандарда кең тараған, алма жемістері мен жапырақтарын зақымдайтын ауру түрі.

Уақытылы өңдеу шаралары жүргізілмеген жағдайда, инфекция алма жемістерінің

сапасы мен алма түсімінің төмендеуіне әкеледі.

Өндірістік бақтарда алма таз қотырының алдын алу үшін маусымына он бес рет

саңырауқұлақтарға қарсы өңдеу жұмыстары жүргізіледі. Соған қарамастан,

фунгицидтер экономикалық шығыны жоғары және экологиялық қауіптеріне

байланысты аурумен күресте оңтайлы жол болып табылмайды. Ауруға төзімді алма

сұрыптарын пайдалану аурумен күресуде тиімдірек және экологиялық таза. Malus

floribunda 821 жабайы түлерінен бөлініп алынған алма таз қотырына резистентті Vf гені

аурудың алдын алу үшін будандастыру бағдарламаларында кеңінен қолданылады.

Ауруға төзімділік гендерімен байланысқан молекулярлық маркерлерді пайдалану

ауруға төзімді сұрыптарды алу үрдісін жылдамдатуға мүмкіндік береді.

Зерттеу жұмысының мақсаты ПТР-ді пайдалана отырып бағаналы алма көшеттеріндегі

Vf генін анықтау. Жас алма көшетінен ДНҚ-ны бөліп алу СТАВ әдісімен жүргізілді. Vf

гені бар сынамалар ПТР әдісінде Vf праймерлері көмегімен электрофорез жасау арқылы

анықталды.

Vf гені алма көшеттерінің төрт сынамасында анықталды. Төрт үлгінің үшеуінде Vf

гендері бағаналы өсумен біріктірілгені анықталды. Бұл зерттеу жұмысының

нәтижесілері ПТР әдісін қолдану алма таз қотырына резистентті Vf генін анықтауда

тиімділігін көрсетеді.

ACKNOWLEDGEMENTS

First of all, I ascribe all glory to the Gracious “Almighty Allah” from whom all blessings

come. I would like to thank Him for His blessing to write this thesis.

I would like to acknowledge my home university Kazakh National Agrarian University and

University of Eastern Finland for giving me the opportunity to study in the Green

Biotechnology and Food Security program, to get more knowledge and to improve my skills.

I would like to express my sincere gratitude to my supervisors for their guidance and support

during the research and thesis writing. I want to say thanks to MSc. Researcher Harri Kokko

for the chance to work in his research group, for sharing his knowledge and experience and for

his guidance in writing and helping with literature survey. I am grateful to MSc Junior

Researcher Anna Toljamo for assisting me during the internship in the laboratories, for

providing me with everything for data collection and analysis.

I want to express special thanks to Roseanna Avento for introducing me to this program and

for the opportunity of being part of it, for guidance and teaching in scientific writing, for

taking care and helping me during difficulties.

In addition, I am very thankful to all UEF staff and teachers who did all their best to teach and

helped us during our stay in Kuopio, Finland.

My enormous thanks to my family and my friends, who always support me in any situations,

who believe in me and for their endless love and friendship. My special thanks go to my friend

Arshad Haroon for his support and guidance during my stay in Kuopio.

This MSc Thesis would not have been possible without the help, patience and support of all

these people mentioned above.

Rakhmet!!!

ABBREVIATIONS AND DEFINITIONS

°C

AFLP

bp

CTAB

DNA

EDTA

Vf

MAS

PCR

PVPP

QTL

RAPD

SCAR

STS

TAE

TE

Celsius degree

Amplified Fragment Length Polymorphism

base pair

Cetyl trimethylammonium bromide

Deoxyribonucleic acid

Ethylenediaminetetraacetic acid

Gene originated from wild apple species Malus floribunda

821 against apple scab caused by Venturia inaequalis

Marker Assisted Selection- a selection for a trait based on

genotype via linked markers instead of the phenotype of the

trait

Polymerase Chain Reaction

Polyvinylpolypyrrolidone

Quantitative Trait Loci

Random Amplified Polymorphic DNA (DNA markers)

Sequence- characterized amplified regions (DNA markers)

Sequence- tagged sites (DNA markers)

Tris-Acetate-EDTA (buffer)

Tris HCl-EDTA (buffer)

CONTENT

ACKNOWLEDGEMENTS ........................................................................................................ 5

1. INTRODUCTION ................................................................................................................ 9

2. LITERATURE REVIEW ................................................................................................... 10

2.1 APPLE ......................................................................................................................... 10

2.1.1 Botany and taxonomy .......................................................................................... 10

2.1.2 History of apple cultivation ................................................................................. 11

2.1.3 Benefits of apple fruit .......................................................................................... 12

2.1.4 Apple production ................................................................................................. 13

2.2 COLUMNAR APPLE TREES .................................................................................... 13

2.3 BREEDING OF APPLE ............................................................................................. 16

2.4 MARKER ASSISTED SELECTION (MAS) ............................................................. 17

2.5 APPLE SCAB ............................................................................................................. 18

2.5.1 Disease History and economic significance ........................................................ 19

2.5.2 Symptoms of apple scab ...................................................................................... 20

2.5.3 Causal Organism ................................................................................................. 21

2.5.4 Life Cycle of the Fungus ..................................................................................... 22

2.5.4.1 Sexual Reproduction .......................................................................................... 22

2.5.4.2 Asexual Reproduction ........................................................................................ 24

2.5.5 Disease Management ........................................................................................... 25

2.6 APPLE SCAB RESISTANCE .................................................................................... 26

2.6.1 Origin of the Vf gene ........................................................................................... 26

2.6.2 Other resistance genes ......................................................................................... 27

2.6.3 Breakdown of apple scab resistance .................................................................... 28

2.6.4 Pyramiding of resistance genes ........................................................................... 29

3. OBJECTIVES .................................................................................................................... 30

4. MATERIALS AND METHODS ....................................................................................... 31

4.1 PLANT MATERIALS ................................................................................................ 31

4.2 DNA EXTRACTION USING CTAB METHOD ....................................................... 32

4.3 PCR USING Vf PRIMERS ......................................................................................... 34

4.4 CLASSIFICATION OF APPLE SEEDLINGS .......................................................... 35

5. RESULTS........................................................................................................................... 36

5.1 DNA EXTRACTION FROM FRESH LEAVES ........................................................ 36

5.2 PCR ANALYSIS TO DETECT Vf GENE .................................................................. 36

5.3 CLASSIFICATION OF APPLE SEEDLINGS .......................................................... 38

6. DISCUSSION .................................................................................................................... 39

7. CONCLUSIONS ................................................................................................................ 41

REFERENCES .......................................................................................................................... 42

9

1. INTRODUCTION

Apple is one of the most common fruit crops in the world. This is due to the extraordinary

diversity of its and many yielding cultivars that have been adapted to the most different soil and

climatic conditions. However, diseases, such as apple scab, may severely hamper apple

production, especially in organic farming (Sandskär, 2003). Apple scab, caused by Venturia

inaequalis, is a widespread disease in apple-growing areas. It attacks fruits, leaves, and reduces

the yield and quality of crop trees. In commercial orchards to prevent or to handle the disease,

fifteen fungicide treatments are required every year (Belfanti et al., 2004).

The Vf gene has been the most widely used resistance gene to apple scab (Gessler and Pertot,

2012). An apple breeding program using monogenic resistance was implemented for the first

time by crossing the wild species Malus floribunda 821, which is the major carrier of the

resistance Vf gene, with other cultivars (Be´naouf and Parisi, 2000). In recent years, new cultivars

and promising donor-seedlings carrying Vf resistance genes have been received.

The transfer of Vf genes by traditional breeding techniques to achieve new resistant apple

cultivars is difficult because it takes effort and time. Besides traditional approaches, DNA

markers are employed in breeding programs. A majority of deoxyribonucleic acid (DNA)

markers of resistance to V. inaequalisis are based on the polymerase chain reaction (PCR) (Vejl

et al., 2003). A number of random amplified polymorphic DNA (RAPD) markers closely linked

to apple scab resistance Vf gene have been found (Tartarini, et al., 1999). As stated by Tartarini et

al. (1999), the use of molecular markers linked to the different resistance genes can noticeably

improve and speed up the whole selection process in juvenile phase of apple seedlings.

The aim of this study was to detect the Vf gene from columnar apple seedlings by using PCR.

10

2. LITERATURE REVIEW

2.1 APPLE

2.1.1 Botany and taxonomy

Apples (genus Malus) belong to the Rosaceae family, in the one of its subfamilies (Maloideae)

like medlars (Mespilus germanica L.), quinces (Cydonia oblonga Mill.), pears (Pyrus L. spp.)

and loquats (Eriobotrya japonica Thunb.) (Turechek, 2004). As reported by Luby (2003), until

the mid of the 19th century most of the studied species of Malus genus were included into Pyrus.

The Malus species have been categorized in variable number of sections, such as Malus,

Sorbomalus, Eriolobus, Choromeles and Docyniopsis, based on their morphological features and

flavonoid analogies, and some of these sections are further divided into series (Luby, 2003; Harris

et al., 2002). Approximately 30 species derived naturally or artificially are known in Malus genus.

Earlier research studies showed that Malus sieversii was the most important source of gene pool

of Malus × domestica (Kumar et al., 2014). However, as Cornille et al. (2014) reported,

European crabapple Malus sylvestris has been an important secondary contributor as it is similar

to domesticated apple species. Malus sieversii has interesting diversity for different important

traits, and thus it can be used in breeding to improve and develop market driven cultivars

(Pereira-Lorenzo et al., 2009; Janick 2005). The Malus taxonomy is challenging as the

differences between wild and cultivated species are unclear and therefore difficult to distinguish

particular varieties (Harris et al., 2002).

Apple is a monoecious species with hermaphroditic flowers. It produces epigynous rose flowers

with five sepals, petals and pistils. The development of several carpellate inferior ovary (forming

the core) and support tissue after fertilization becomes the fruit, known as pome. Nearly all apple

cultivars are self-incompatible and for the fertilization and to develop fruits, apple flowers need

to be cross-pollinated (Kumar et al., 2014; Sandskär, 2003). Generally, most of the cultivated

apples are diploids (2n=34), and during the meiosis 17 bivalents are formed, even though there

exist certain triploid (3x=51) cultivars, for example ‘Ribston Pippin’, ‘Bramley’ and ‘Jonagold

(Pereira-Lorenzo et al, 2009).

11

Owing to polymorphism, apple has extraordinary diversity. Depending on varieties, apple fruits

can differ in colors and shades, size with oval or pear shapes. Nowadays there are more than

10000 varieties of apple, which vary in taste, shape, juiciness, texture, color, firmness and other

qualities.

2.1.2 History of apple cultivation

The native species of genus Malus originated from the region of Asia Minor, Central Asia, the

Caucasus, China, Pakistan and Himalayan India. The opening of trade routes from China to the

Middle East and Europe via Central Asia – “The Old Silk Road” played a key role in the

evolution and in spreading of cultivated apple (Boudichevskaja, 2009; Forsline et al., 2003).

As reported by Boudichevskaja (2009) in Ancient Greece and Persia, certain techniques (pruning,

storage, grafting, precise rootstock usage, and selection of distinctive apple clones) of apple

cultivation were already used and twenty varieties of cultivated apples in several regions were

described. Janick (2005) mentioned that Persia was patently the source of apple and other

numerous fruits originated from Central Asia.

Wild apple fruits were cultivated and frequently spread throughout the Europe and Mideast,

which led to the cultivation of thousands of unique and new varieties (Janick, 2005). Apples have

been widely planted all over Europe and consumed as a raw fruit, fermented juice or cider,

vinegar, applejack as well as cooked and mixed with sugar and sweets. In the 18th and 19th

centuries, more than one hundred apple cultivars were described and categorized based on their

final application and availability (Harris et al., 2002). Until the introduction of M. × domestica in

the late 19th century, the Chinese pear leaf crabapple Malus asiatica Nakai was the major

cultivated species in southern and eastern Asia. After M. domestica was introduced, breeding

started in many parts of the world and the commercial apple production existing todays began

(Luby, 2003).

12

2.1.3 Benefits of apple fruit

Apple is considered as a significant provider for human well-being as a valuable source of

phytonutrients and natural antioxidants, as well as important minerals. In addition, apples are rich

in beta-carotene and B-complex vitamins, saccharides, pectin and fiber (Boyer et al., 2004).

Apples have the low concentration of calorie, fat and fatty acids, sodium (Volk et al., 2014). All

compounds in apple are necessary for the normal development and growth (Zhang et al., 2014).

Apples are also an important raw material used for fresh consumption and for many fields of

processing industry (Vejl et.al., 2003). The main nutrient values of apple can be seen in Table 1.

Table1. Average nutrient content in apple (per 100 g fresh weight)

Nutrients Nutrient value In percentage

Energy 52 Kcal 2.5%

Carbohydrates 13.81 g 11%

Protein 0.26 g 0.5%

Total Fat 0.17 g 0.5%

Dietary Fiber 2.40 g 6%

Pantothenic acid 0.061 mg 1%

Vitamin C 4.6 mg 8%

Sodium 1 mg 0%

Potassium 107 mg 2%

Calcium 6 mg 0.6%

Iron 0.12 mg 1%

Carotene-ß 27 µg --

(Source USAD, 2016)

Phytochemicals, especially flavonoids in apple have a protective effect against oxidation and

reduce accumulation of oxygen in tissues (Volk et al., 2014). Moreover, Ferretti et al. (2014)

claim that the level of antioxidant activity in apple varieties is higher in comparison to other fruits

and vegetables. Besides, in addition of fiber and antioxidants to human body, apple consumption

was linked with decreased the risk of cancer (lung cancer), asthma as well as diabetes (Ferretti et

al. 2014; Hyson, 2011; Boyer et al., 2004).

13

2.1.4 Apple production

Nowadays as an important commercial crop, apple is cultivated in many parts of the world

(Kellerhals, 2009; Pereira-Lorenzo et al., 2009). Indeed, apple is the most widely spread

important and consumed species of temperate fruit crops grown in high-latitude regions (Forsline

et al., 2003).

For the last twenty years, apple production has expanded more than fifty percent and the biggest

growth has been in China (Brown, 2012). China as the largest apple producer in the world

commercially produces 35 million tons of apples followed by the production in the United States

of America (USA) of 4.2 million tons (Volk et al., 2014; Kellarhals, 2009). In the USA, apple is

the third most valued fruit crop after grapes and oranges. Volk et al. (2014) reported that

production of apple in the USA is ranked 17th for its production value. Apple products including

canned apple and extracted juices make about thirty-three percent of the apple crop in the USA.

Currently, some cultivars, such as Delicious and Golden Delicious are grown almost in all apple-

producing countries. In the major countries, which produce apple, breeding projects are

producing new cultivars with enhanced quality and other features, for instance, disease resistance

(Jones and Aldwinkle, 1990).

2.2 COLUMNAR APPLE TREES

Today, columnar apple trees are becoming more commonly used shapes and methods of growing

cultivars. These apple cultivars are upright growing cylindrical trees with a terminal and fruiting

spurs along the main leader that bear the fruit (Figure 1). Columnar apple cultivars have a thick

stem with fewer differences in diameters on the base and top, short branches and internodes,

reduced height, compared to apple trees, with normal growth habit (Otto et al., 2013).

In addition, there are differences in leaves between columnar and normal apple trees. Columnar

apple varieties have dark green coloured and notched leaves with long petioles (Petersen and

Krost, 2013). Morimoto and Banno (2014) reported that columnar apple trees can be described by

differences in leaf thickness, shape and leaf area. Clear detection of columnar apples at the early

14

stage of growth is often erroneous, due to the existence of various intermediary types as well as

numerous growth phenotypes, but after two or three years, obvious corroboration of columnar

growth habit can be done.

Figure 1. Columnar apple tree from orchard of Ruokamarja Oy, Pellesmäki, Kuopio, Finland

(Photograph: Harri Kokko)

Initially, in the 1960s the columnar growth habit in apple cultivars was first acquired as a

columnar mutation of the McIntosh in East Malling Research (EMR) in the UK and in honour of

a researcher called “Wijcik McIntosh” (Otto et al., 2013; Dokoupil and Řezníček, 2012; Bai et

al., 2012). Apple cultivars with columnar growth habit were one of the recent purposes of apple

breeding programs for the genetic enhancement in apple tree architecture to produce new apple

cultivars with more compact growth habit (Bai et al., 2012; Moriya et al., 2009). Until the

twentieth century, after many crossing processes more than hundred different apple cultivars have

been released from Wijcik McIntosh, however, most of these cultivars were not of marketable

quality and were also susceptible to diseases (Blažek and Křelinová, 2011).

15

Columnar varieties achieved from breeding approaches in certain countries such as Belgium,

Korea, the USA, Germany, Latvia (Ikase and Dumbravs, 2004) were higher in fruit quality and

resistant to widespread diseases like fire bright, powdery mildew and apple scab (Petersen and

Krost, 2013; Dokoupil and Řezníček, 2012). Several factors as genotype, adaptation to the certain

climate and region, rootstock, have influence to the growth, yielding fruit quality of these apple

cultivars (Ikase, 2007).

These columnar apple cultivars are becoming widespread, particularly among small-scale

gardeners, due to the ease of cultivating them in small gardens. They require minimal pruning

without any additional manual labor and cost, thus providing economic benefits (Otto et al.,

2013; Dokoupil and Řezníček, 2012). Pruning is most often concentrated only on cutting off

shoots once in a year usually in summer. Using genotypes with a spontaneous regulation of fruit

setting has a favorable effect on the quality of fruit on the surface parts of the crown. The costs

for individual support will be reduced by using suitable rootstock if the tree is firmly anchored in

the soil (Dokoupil and Řezníček, 2012). Most of the columnar apple cultivars are resistant to

drought and cold.

The dominant allele termed Columnar (Co) gene, with one or more modifiers involved, is the

main contributing gene, which stimulates the major features of tree architecture. This gene, which

occurs in a heterozygous state, is set on chromosome 10. Currently, the characteristics and role of

Co gene and its mutation type remain to be unidentified and thus it is not well established

(Petersen and Krost, 2013; Wolters et al., 2013; Ikase and Dumbravs, 2004). Furthermore,

several investigations (Petersen and Krost, 2013; Layne and Bassi, 2008) found out that other

Rosaceae species like cherry and peach have shown the columnar growth phenotype too.

Krost et al. (2012) stated that the involvement of different levels of growth regulating hormones

like cytokinins, gibberellic acid (GA), indoleacetic acid (IAA) and abscisic acid (ABA) seems to

be comprehensible on the columnar phenotype. As these cultivars have short lateral branches and

fruit spurs, the main attention is assumed on the level of auxin, which can effect to the apical

control. It seems that levels of abscisic acid and gibberellin are low whereas the ratio of

auxin/cytokinin is high in columnar apple. In the columnar compared to common apples, the

level of cytokinines is higher and cytokine metabolism is altered (Petersen and Krost, 2013).

16

2.3 BREEDING OF APPLE

Apple breeding is usually a long process and expensive. Spanned over an average time of twenty

years, the production cycle goes from primary crossings (interbreeding, backcrossing, grafting,

etc.) to the cultivation of apple commercially (Sandskär, 2003). Traditional apple breeding

strategies are associated with crossing in the middle of leading commercial and elite cultivars, as

well as planting for clone analysis. The juvenile period in apple grown in orchards is extended.

Flowering may start after four years, fruiting in six years, but in some cases, this period may last

more than ten years. However, by using flower bud-inducing practices and boosting the growth

of seedlings in greenhouses, the juvenile phase can be shortened (Kumar et al., 2014).

The majority of breeding programs are globally found on narrow genetic pools and can vary due

to different objectives. Numerous important characteristics such as winter hardiness, harvesting

period, growth habit, resistance to pest and diseases, also fruit qualities (shape, size, acidity,

juiciness, sweetness, color) and other features are primary objectives in most apple breeding

programs worldwide (Kellerhals, 2009; Sansavini et al., 2004).

Depending on the weather conditions of apple-growing regions, breeding objectives can vary, for

example, flower intensity with high bud break are the main priority in regions with mild winter

conditions, whereas cold endurance is the more important aim in regions with harsh weather in

spring, winter and fall (Kumar et al., 2014). High and reliable productivity and adaptability to

different weather conditions are the important requirements for commercial apple cultivars.

Breeders aim at genetic enhancement in preserving flesh texture, for the duration of permanent

cold storage, by reducing storage periods and storage defects.

In most apple breeding programs, resistance to diseases and distinct pests, in several areas,

remains to be a relevant substantive target (Kumar et al., 2014). Pathogen and pest resistance to

certain chemicals is a persistent problem for the apple industry. In addition, consumers are

worried about environmental sustainability and the safety of food products. The resistance of

plants offers a cheaper and more lasting solution for the protection of trees and fruits against

damages. In cases when there is no appropriate chemical control of diseases, the development of

resistant varieties is the sole way for efficient apple production (Kumar et al., 2014).

17

Breeding of apple for the resistance to diseases is of high importance especially for organic

cultivars (Sandskär, 2003). Resistance to diseases includes prevention against more than just one

disease. The apple crops resistant to apple scab are still vulnerable to other diseases such as

powdery mildew (Brown, 2012).

2.4 MARKER ASSISTED SELECTION (MAS)

In the last decade, much effort has been expended in basic research to explore the genetic basis of

a given trait and to identify markers genetically linked to it (Sansavini et al., 2004). With the

advancement in the field of molecular biology, the usage of molecular markers has been

introduced in apple breeding and many markers associated with monogenic traits have been

distinguished (Pătraşcu et al., 2006). These techniques have proved to be effective in terms of

accelerating and better breeding of apple (Kumar et al., 2014; Pătraşcu et al., 2006). They

increase the accuracy of selection process and enable the pyramiding of several resistance genes.

Several breeding programs in the USA and France, employ MAS especially for parental selection

for loci that include fruit color, acidity, crispness, Vf gene and Quantitative Trait Loci (QTL)

resistances to apple scab (Kumar et al., 2014, Kellarhals, 2009).

Generally, the molecular marker techniques involve the investigation of plants at the seedling

phase by the inspection of plant DNA. The specific DNA molecular markers characterize genetic

traits among individual organisms or species. Usually, these markers work like “signs” that

represent the genes themselves and do not have any effect on the phenotype since they are

detected near the genes that control the feature of interest. Other than DNA markers,

morphological and biochemical markers, were particularly beneficial for plant breeders, however,

they are affected by the growing stage of the plant and environment and are confined in quantity

(Ignatov and Bodishevskaya, 2011).

Specifically, some DNA markers such as random amplified polymorphic DNA (RAPDs) are

prone to experimental factors which result in weak reproducibility and reliability (Li et al., 2012).

The Amplified Fragment Length Polymorphism (AFLP) markers have been commonly applied

for genetic mapping, confirmation of plant characteristics and assessment of genetic diversity

18

(Brumlop and Finckh, 2011). AFLP technique associates with the productivity of PCR-based

markers like RAPD, but is considered more reliable. However, AFLP markers are dominant, and

differentiation between homo- and heterozygous genotypes is not possible.

The main disadvantages of RAPD and AFLP markers led to the improvement of new more

advanced markers by cloning and sequencing them, such as sequence- tagged sites (STS) or

sequence- characterized amplified regions (SCAR) markers (Ignatov and Bodishevskaya, 2011).

SCAR markers may be codominant and they identify a single locus. The transformation of

RAPDs into SCARs also yields good results in dealing with the powdery mildew

(Boudichevskaja, 2009).

Through the ability to detect pyramidization of multiple resistance sources and complex genetic

combinations, PCR based markers, SCAR and SSR markers become valuable in breeding

programs linked to useful traits for crop plants especially to identify apple tree cultivars. In

addition, these markers can be integrated with multiplex PCR reactions and analyzed on

automatic sequencers with short running times (Ignatov and Bodishevskaya, 2011).

2.5 APPLE SCAB

Apples are susceptible to more than seventy diseases caused by bacteria, viruses and by

pathogenic fungi. Generally, the outcome of the combination of numerous techniques of disease

control is the conquering managing of the disease. Using resistant rootstocks and grafts,

bactericides, fungicides, improve the environment and spot selection are among the tools used to

control apple diseases. In comparison to other plant pathogens, pathogenic fungi can be the

reason of many disorders and diseases like fruit and leaf spot, defoliation and decomposition,

damages of leaf, blossom and different types of cancers (Grove and Xiao, 2005).

19

2.5.1 Disease History and economic significance

Apple scab or black spot is a destructive disease that is caused by pathogen fungus Venturia

inaequalis (Cke.) Winter (fungus was first called Spilocaea pomi), which also can infect

firethorn, hawthorn and crabapple (Jha et al., 2009). There is no exact information when scab was

found first time in orchards. Swedish botanist Fries, in 1819, gave the first description of the

disease, but the oldest data of its existence was presented in the painting of sixteenth century

(MacHardy et al. 2001). The occurrence of scab in the eastern United States was discovered at

the beginning of 1834 (Ogawa et al., 1991).

Nowadays, apple scab is an economically most significant disease, especially, in temperate apple

growing regions with cool, wet weather in spring and high summer rainfall. The uncontrolled

disease may result in the reduction of fruit quality and quantity or almost complete devastation of

an apple crop. Apple scab can be detected on petioles, leaves, sepals, fruits and seldom in young

shoots and bud scales (Vaillancourt and Hartman, 2000). Infection of fruits and pedicel is the

main reason for the direct losses (Ogawa et al., 1991).

Heavy leaf infection followed by defoliation is observed on susceptible apple cultivars. Other

debilitating effects of scab are the low viability of apple trees, increased susceptibility to winter

injury, reduced formation of leaf buds and fruits, lower growth and reduced yields in the next

years (Jamar, 2011). If the scab is chemically controlled, losses can be minimized by spraying

with the fungicides, but the production costs increase together with increasing health and

ecological concerns (Pătraşcu et al., 2006). Scab can develop and cause fruit rot during storage

due to low vitamin concentration, resulting in significant losses (Jamar, 2011). Various factors

influence the speed of disease and final disease complication and seriousness, including

sanitation, cultivar susceptibility and frequency of the infection periods (Jones and Aldwinkle,

1990).

20

2.5.2 Symptoms of apple scab

Generally, the most visible and severe symptoms occur on leaves and fruits (Daniels, 2013;

Giraud et al., 2011; Sandskär, 2003). Different apple cultivars show particular leaf symptoms

very differently. The earliest spring lesions of scab are clearly defined dark green velvety spots,

which commonly appear on the lower surfaces of expanding leaves (Figure 2) (Giraud et al.,

2011; Turechek, 2004). The circular lesions develop mostly on the upper surfaces of developed

leaves and increase in size, consequently splintered, swelled and scaly leaves turn yellow and fall

on the ground (Vaillancourt and Hartman, 2000). Severity of infection depends on the age of

leaves and fruits. Especially young tissues can be infected easily but after expanding they become

more resistant (Li and Xu, 2002). After inoculation, scab attacks reduce the activity of

photosynthesis apparatus, assimilation of CO2 and water potential, the formation of leaves and

setting of fruit buds. Infected tissues may crumple and become distorted (Ogawa et al., 1991).

After petal fall, fruits are extremely vulnerable to infection during long humid periods. The

visible symptoms on young fruit occur as green to olive-brown lesions close to the blossom end

and microorganisms, entered via cracks, may cause fruit decays and drop from the tree (Sharma,

2005). As shown in Figure 2, fruit lesions cover apple skin with black spots that expand and

produce scabby patches and develop into necrotic. During the growth period in summer, wet

weather can also affect the development of scab. Symptoms of infection can appear on sepals

after the fruit development because of lesions on the fruit sides (Turechek, 2004).

Jha et al. (2009) stated that symptoms of scab infection emerged on twigs of some apple cultivars

occur as light brown swellings surrounded by whitish rings. Sharma (2005) described twig

infections as small reddish-brown dots and peeling with the dark green mass under it. Some

infected apple cultivars may have reddish brown colored small lesions developed on the twigs

and during bloom dark green lesions on the sepals, at the base of the flower (Ogawa et al., 1991).

Weakened trees, which are susceptible to various diseases, insects and freeze damage, is a

consequence of a significant defoliation over several years (Vaillancourt and Hartman, 2000).

21

Figure 2. Apple scab symptoms on leaves and fruits from orchard of Ruokamarja Oy, Pellesmäki,

Kuopio, Finland (Photograph: Harri Kokko)

2.5.3 Causal Organism

Venturia inaequalis (Cke) Winter is the causal organism of apple scab that belongs to genus

Venturia (Jamar, 2011; Sandskär, 2003). MacHardy (1996) has studied its taxonomy and he

classified it to the subdivision Ascomycota, class Loculoascomyctes, order Pleosporales and

family Venturiaceae. Two different states of this fungus are saprophytic or the perfect (sexual)

state Venturia inaequalis (Cke) Winter, and the parasitic or imperfect (asexual) state Spilocaea

pomi Fr. (Jamar, 2011; Bowen et al., 2011). Fundamentally, V. inaequalis only infects Malus

species. However, there are other members of the ascomycete fungus detected in New Zealand,

Canada, which cause scab in other crop fruits, for example, V. carpophila infects peach and V.

pirina causes pear scab (Ogawa et al., 1991).

V. inaequalis was one of the first studied ascomycetes and remains to be a practical

implementation for numerous genetic studies, for example, its sexual compatibility and the

heritability of pathogenicity. This is caused by similarity to other parasites that infect young

living tissues without obvious damage for a long period as well as its ability to be cultivated and

mate in vitro (Vaillancourt and Hartman, 2005). Among the characteristics that make V.

inaequalis so acceptable to genetic studies include its genotype and phenotype stability for many

years and large diversity in nature. Living tissues are infected by heterothallic fungus V.

inaequalis (Jha, et al., 2009).

22

The bio-ecology of V. inaequalis has been studied extensively in several countries. Susceptibility

of apple cultivar, pathogen inoculation, growing phase of the tree, the maturity of fruits,

discharge of ascospores (sexual spores), and abiotic factors such as light or relative humidity, air

temperature, duration of leaf wetness influence the spread of infection risks of apple trees

(Bowen et al., 2011; Raudonis et al., 2008). The degree of resistance in some commercial

genotypes changes over time due to a particular fungus adaptation to plant host and even in some

wild Malus species or genotypes resistance can be disrupted by new races (Jha et al., 2009). The

presence of several races of V. inaequalis, which occur in the USA and Japan, makes it

significantly difficult to breed resistant apple cultivars.

2.5.4 Life Cycle of the Fungus

The fungus that causes the disease overwinters mostly in dead leaves, in which microscopic

flask-formed black fruiting bodies, called pseudothecia, are developed. In the early spring, the

ascospores inside pseudothecia start to mature and in suitable weather conditions, when leaves on

the orchard become wet after the rain, spores are forcibly ejected into the air (Jamar, 2011;

Sandskär, 2003). A detailed life cycle of apple scab disease is shown in Figure 3.

2.5.4.1 Sexual Reproduction

For sexual reproduction, the presence of two mating types is essential. In the beginning of spring,

mating proceeds in infected leaves from the prior season. During winter and early spring, fungus

forms fruiting bodies called pseudothecium. Pseudothecium develops in the fallen leaves after the

fertilization. Formed pseudothecium contains sacs, which are termed asci, and each elongated and

cylindrical ascus has eight ascospores (11-15 µm long and 4-8 µm wide). Asci are bitunicate,

cylinder-shaped, double walled (Jha et al., 2009).

Ascomycete fungus, like other parasites, infects the living tissues by producing ascospores

(Daniels, 2013). Ascospores are sexual spores produced in the pseudothecia and function as an

initial source of inoculum. Released fungus colonies are velvety and dark olivaceous brown.

Brown colored ascospores have two cells with different shapes, from where the Latin name of the

fungus “inaequalis” is inferred (Vaillancourt and Hartman, 2000).

23

Figure 3. Life cycle of apple scab disease caused by Venturia inaequalis (Biggs and Stensvand, 2014)

The development of ascospores continues during the spring and some spores mature and start to

increase quickly while the bud breaks and blossom period occur when there are favorable

conditions. The ascospores are passively emitted into the air, when the rain wets the fallen leaves

(Raudonis et al., 2008).

24

For optimal ascosporic release, the presence of light, high humidity and leaf wetness are

required. Developed pseudothecia distend and stick out from the surface of the leaf. Unrestricted

ascospores are transferred by rain and wind into the air and when landed on young blossoms,

fruitlets, and leaves, ascospores germinate and start to infect the apple tree (Sandskär, 2003). Due

to temperature, time for the infection period varies and the first symptoms may appear

approximately after two weeks. The symptoms of primary infection are velvety or olive-brown

spots and dark conidia referred to as summer spots occur like bear masses. One cycle of the

infection occurs in one season (Vaillancourt and Hartman, 2000).

2.5.4.2 Asexual Reproduction

According to Vaillancourt and Hartman (2000), asexual reproduction of V. inaequalis starts by

producing conidia. Spilocaea pomi is known as the conidial stage of the V. inaequalis. The

conidia are olive or brown colored single-cells with width of 6-12 µm and length of 12-22 µm.

They are produced one after the other at the tip of short hyphae termed as conidiophores. The

conidia and conidiophores give a distinctive velvety exterior to the newly developed lesions of

scab as mass produced on the thick mat of mycelium.

Once distributed by the wind and flopping rain, conidia land on an apple blossom or fruit and

leaves, and stick to the surface and germinate. The hyphae germination breaks through the cuticle

and creates a new infection. The conidia of V. inaequalis are able to adhere and germinate also on

non-host plants. Like in the case of ascospores, the discharging of conidia depends on the

temperature as well as moisture and humidity, and may develop from few days to a couple of

weeks after initial leaf infection. The perfect conditions for the development of secondary

infection by conidia are wet and cool days in spring, summer and fall (Biggs and Stensvand,

2014). Many cycles of conidial production and secondary infection take place in a particular

growing period under the suitable weather conditions. Late infection in autumn may not be

detected. However, during storage, it can affect fruits (Sandskär, 2003).

25

2.5.5 Disease Management

After intensive apple production over the world, apple scab has become a serious problem for

large scaled orchards. At the end of 19th century first chemical fungicides, especially protective

fungicides based on copper against pathogen V. inaequalis, were discovered. Recently based on

epidemiological trainings, mineral fungicides like sulfur with less toxicity were developed. In

addition, in the 20th century, using of synthetic organic fungicides improved curative fungicidal

control (Jamar, 2011). Farmers have to treat apple cultivars, which are vulnerable for scab,

twenty-thirty times with fungicides per season (Soriano et al., 2009). Also in organic apple

production, applications of mineral substances like sulfur, lime sulfur, and copper salts are

essential for effective scab management to preserve the resistance of cultivars and to prevent

initial infection (Daniels, 2013; Beckerman, 2009).

Initial infection produced by ascospores is the main object, on which chemical control of apple

scab is concentrated. Later fungicide sprays are targeted at other fungal diseases as well as

against secondary infection of apple scab. The strategies of using fungicides are formed on the

base of studies of apple scab epidemiology and biology throughout the years (Cova et al., 2015;

Alaniz et al., 2014). In general, growers apply preventive and curative chemical fungicides

together for good efficiency that are capable of stopping fungus development (Chapman et al.,

2011; Vaillancourt and Hartman, 2005). Currently, to reduce fungicide applications, enhanced

strategies are developed based on a better understanding of the airborne spreading of ascospores,

host foliage and fruit area, the host defenselessness against infection (Aylor, 1998).

In addition, standard cultural and sanitary practices are used to reduce scab infection such as leaf

shredding, burning or burying leaves in the soil, and urea treatments, which help to discourage

the development of V. inaequalis (Giraud et al., 2011). Regular pruning is needed for the proper

sunlight penetration and air movement between trees for the prevention of scab development.

Biological control, such as utilizing natural microbial antagonists to V. inaequalis,

microorganisms to confer resistance to V. inaequalis infections, or to break down apple leaf litter

in autumn, can be a practical way to control apple scab in the future (Jamar, 2011; Vaillancourt

and Hartman, 2005).

26

2.6 APPLE SCAB RESISTANCE

Resistance genes are set of genes developed in plants to identify and produce resistance reactions

against pathogens. The pathogenic factors detected by these genes are termed avirulence factors

for the reason that they make the pathogen avirulent. A number of resistant gene containing loci

have been isolated from apple cultivars (Manafu et al., 2014). These resistance genes can be

classified according to the level of resistant.

As depicted by Jamar (2009) resistance can be of different types:

vertical resistance and horizontal resistance;

depending on the number of genes that controls the resistance (monogenic- by single

gene, oligogenic- by small number of genes or one major gene along with a few minor

genes, and polygenic resistance - controlled by a set of minor genes) (Gessler et al.,

2006);

depending on the host and pathogen interaction (total resistance or immunity and partial

resistance) (Bastiaanse et al., 2015);

ontogenic resistance (associated with the leaf age) (Alaniz et al., 2014);

durable resistance (remains effective for a long period) (Broggini et al., 2011).

2.6.1 Origin of the Vf gene

Scab has become a disease requiring severe chemical control programs that are expensive, bulky

and polluting. Researchers have accepted the significance of breeding for resistance at an early

stage and the benefits of introgression resistance from wild Malus species (Gessler and Pertot,

2012). The Vf locus originated from wild apple species Malus floribunda 821 is an important

source of resistance to apple scab disease caused by V. inaequalis. It has been introduced into

various domesticated apple cultivars (Afunian et al., 2004).

Researchers of the University of Illinois crossed a generous amount of commercial cultivars

with crab apples (Gessler and Pertot, 2012; Janick, 2002). Two siblings (F226829-2-2 and

F226830-2) which were distinguished as scab resistant after several crosses, were applied in

further crosses as well as were the starting material for the PRI Cooperation Program (Purdue

27

University (IN), Rutgers University (NJ) and the University of Illinois) (Gessler and Pertot,

2012). The inheritance of scab resistance was analyzed and called Vf (the first letter refers to as

the name of the causal pathogen Venturia and the second letter refers to as the name of apple

species, from which it was originated M. floribunda) (Pătraşcu et al., 2006). The first cultivar

with acceptable quality and fruit size derived in the long series of PRI program was apple

cultivar ‘Prima’ (Gessler and Pertot, 2012, Vejl et al., 2003).

Many attempts were made to breed scab resistance around the world between the 1970s and

1980s. Several research works were carried out in the extensive efforts to study the nature

along with the stability of resistance. The selection of disease-resistant cultivars remains to be a

priority for all major Western breeding programs and most of them have been using mainly Vf

resistance. Various Malus species and hybrids having genetic resistance have been

distinguished and currently, above eighty cultivars have been released and named (Gessler and

Pertot, 2012; Pătraşcu et al., 2006). However, the market share of scab-resistance cultivars has

been small, even though they were found acceptable in organic production.

2.6.2 Other resistance genes

Besides the Vf gene, there are several other scab resistance genes that can be used in apple

breeding programs such as Va (from the Antonovka PI 172623), Vb (from M. baccata Hansen’s

no. 2), Vbj (from M. baccata var. jackii), Vm (from M. micromalus) and Vr2 (from ‘GMAL 2473)

(Bus et al., 2011; Jamar, 2011; Gessler et al., 2006). Marić et al. (2010) have reported that fifteen

resistance genes against apple scab have been detected. These genes have been known for long

period, however, only two cultivars (‘Murray’ and ‘Rouville’) carrying Vm gene have been

released so far (Cova et al., 2015). The Vg gene, which gives resistance to ‘Golden Delicious’

and some of its progeny, was identified by Be´naouf and Parisi (2000). Later, more scab

resistance genes (Vh2, Vh4, Vd and Vr2) were determined and mapped (Jha et al., 2009). Two

other cultivars have also been bred with these resistance genes (‘Durello di Forlì’, carrying Vd;

‘Regia’ carrying Vh4) (Gessler and Pertot, 2012; Soriano et al., 2009; Pătraşcu et al., 2006).

28

2.6.3 Breakdown of apple scab resistance

It was observed in several investigations (Jha et al., 2009; Soriano et al., 2009) that the widely

used resistance Vf gene originated from M. floribunda 821 was overcome by some V. inaequalis

races. As referred by Bus et al. (2011) and Jamar (2011), the term ‘race’ for V. inaequalis defines

an isolate, which is able to infect and ultimately sporulate on a specific host resistant to other

isolates; as referred to in the previous literature it should be termed as physiological race. In the

case of V. inaequalis, which is the obligatory sexually reproducing; this term designates the

presence or lack of virulence traits to particular hosts (Gessler et al., 2006). Presently, according

to the virulence of V. inaequalis on ‘specific host’ varieties eight physiological races of scab are

defined (Jamar, 2011). In Table 2, seven different races of V. inaequalis are described.

Table 2. Races of V.inaequalis and Differential hosts of apple species and their sources

Races Susceptible cultivars Resistant cultivars Source

1 Popular domestic apple cultivars Malus clones ‘Dolgo’,

R12740-7A and ‘Geneva’

Worldwide

2 ‘Dolgo,’ ‘Geneva’ and certain

offspring of ‘R12740-7A

‘Florina’, M.floribunda 821 South Dakota, US

‘Geneva’, ‘Golden Delicious’ ‘Florina’, M.floribunda 821,

‘Dolgo’

Nova Scotia,

Canada

4 Offspring of ‘R12740-7A’ ‘Dolgo’, ‘Geneva’, ‘Florina’,

M.floribunda 821

Lafayette,

Indiana, Purdue,

US

5 Carriers of the Vm resistance and

Malus micromalus

‘Dolgo’, ‘Geneva’, ‘Florina’,

M.floribunda 821

Norwich,

England

6 Most varieties containing the Vf

gene, ‘Prima’

M. floribunda 821, the

ornamental crabapple (Malus ×

Perpetu) ‘Evereste’ (Vbj, Vr

and Va)

Ahrensburg,

Germany

7 Malus floribunda 821 and Vf

cultivars ‘Jonafree’, ‘Liberty’,

‘Redfree’

‘Golden Delicious’ (Vg) and

certain varieties that carry the

Vf gene, such as ‘Prima’

East Malling in

the UK. Europe

(Data from Jamar, 2011; Jha et al., 2009; Sandskär, 2003, Janick, 2002)

29

Races 6 and 7 carry the Vf gene. Race 6 was identified in Ahrensburg, Germany, which gave

symptoms on some cultivars carrying Vf gene descended from M. floribunda 821 while, cultivars

with other resistance types (Va, Vr, Vbj) remained uninfected (Parisi et al., 1993). Lately, M.

floribunda 821 was attacked by race 7, which spreads faster compared to race 6 (Xu et al., 2009;

Guérin and Le Cam, 2004). The detection of races 6 and 7 confirmed that all Vf orchards can be

susceptible to V. inaequalis (Daniels, 2013). However, in Northern Europe the Vf resistance is

still considered as valuable protection against scab.

2.6.4 Pyramiding of resistance genes

Presently, the resistance is only due to Vf gene in most scab-resistant cultivars. Some breeders

have cautioned that extensive use of Vf gene can escalate the risk of selection for pathogen

genotypes, which are capable to surmount this resistance (Gessler and Pertot, 2012). As

suggested by Soriano et al. (2009) a number of resistance genes should be piled up (pyramiding)

for durable resistance. In breeding programs, the pyramiding of several resistance genes is an

important strategy, attributable to the genetic mutability of pathogen and the risk of loss of

resistance in apple cultivars growing extensively (Daniels, 2013). By using molecular markers

linked to resistance genes, the selection of cultivars with multiple resistance genes will be

possible (Király et al., 2009; Collard and Mackill, 2008).

30

3. OBJECTIVES

The main aim of this study was to detect the apple scab resistant Vf gene from columnar apple

seedlings by using PCR. Analyzed apple seedlings were classified into groups of columnar and

non-columnar growth type, which have Vf gene.

The hypothesis of this study was that PCR process is a useful way to detect the apple scab

resistant (Vf gene).

31

4. MATERIALS AND METHODS

In this study, the Vf resistance gene was identified from apple seedlings with following

procedure. First, samples were collected from potential columnar apple seedlings. Using Cetyl

trimethylammonium bromide (CTAB) method, DNA extraction was done from fresh leaf

samples. DNA extraction was followed by PCR amplification using pair of Vf primers linked to

Vf gene. In autumn, growth habit of apple seedlings was evaluated again and seedlings were

classified into groups of columnar, potential columnar and normal growth type. The experiments

were done in the laboratories of the Department of Biology at the the Kuopio Campus of the

University of Eastern Finland.

4.1 PLANT MATERIALS

Approximately 200 seedlings of different apple cultivars (Dialog, Dzin, Ikaza, Medok, President,

Valjuta, Vasjugan and X2) were planted into new containers (10 × 10 × 11 cm) in the mixture of

sand, soil and vermiculite (20:75:5) at the end of May 2015. Repotted seedlings were placed in

the greenhouse in controlled growing conditions and they were watered and fertilized daily with

0.1% Ferticare hydro (Yara) (Figure 4).

Leaf samples (n=103) for DNA extraction were collected from potential columnar-type seedlings

and seedlings from Snellmania indoor garden in the middle of June, July and August. For more

reliable identification of columnar growth type, tips of apple seedlings were cut at the beginning

of the summer. If the seedlings were columnar, the new stem was supposed to grow straight and

in parallel to the main stem from the place where tips were cut. Young leaves were chosen,

because they are rich sources of DNA. Leaf disks with 8 mm diameter were cut by punching the

leaves with the cap of micro centrifuge tube. In total, leaf samples were collected from 103 apple

seedlings.

32

Figure 4. Repotted apple seedlings in the UEF greenhouse

4.2 DNA EXTRACTION USING CTAB METHOD

CTAB is used as detergent disintegrating membranes to separate DNA from proteins and lipids.

DNA extraction from the leaves was performed using CTAB method (Doyle and Doyle, 1987) in

the following manner:

First, fresh leaf samples (n-103) were homogenized with TissueLyser in 2 ml micro centrifuge

tubes for 2 minutes at 30 Hz. 750 µl of preheated (60°C) CTAB isolation buffer (2% (w/v)

CTAB, 1.4 M NaCl, 20 mM EDTA, 100 mM Tris-HCl (pH 8.0), 2% (v/v) 2-mercaptoethanol,

1% PVPP) was added to each homogenized sample and they were incubated at 60°C for 30

minutes mixing occasionally. The purpose of these steps was to break down the plant tissue and

release the DNA from the cells.

33

To remove proteins and lipids, samples were extracted once with 750 µl chloroform-isoamyl

alcohol (24:1; V: V) and centrifuged for 10 min at 1700 x g at room temperature. The upper

aqueous phase, containing the DNA, was transferred to a new tube, 500 µl of cold isopropanol

was added and mixed by inverting the tube. Then samples were placed in freezer, overnight, to

precipitate the DNA.

In next step, samples were centrifuged for 10 min at 1700 x g. The supernatant was removed and

the DNA pellet was washed with 1 ml of wash buffer (76% (v/v) ethanol, 10mM ammonium

acetate) for at least 20 min. This step was carried out twice. Briefly dried DNA pellet was re-

suspended in 150 µl TE buffer (pH 8.0) (10mM Tris-HCl (pH 7.4), 1mM EDTA), then 1.5 µl of

RNase A (100 mg/ml) was added and samples were incubated at 37 °C for 30 min to degrade the

RNA molecules. After incubation, tubes were placed on ice and 150 µl of TE buffer, 150 µl of

ammonium acetate (7.5 M) and 1.125 ml of ice-cold ethanol was added. Then DNA was allowed

to precipitate in freezer (- 20 °C) for at least 20 min (or overnight).

To collect the DNA, samples were centrifuged for 10 min at 10 000 x g, supernatant was

removed and DNAs were washed with 1 ml of 80 % ethanol and centrifuged for 10 min at 10 000

x g for a second time. Once the ethanol was removed the DNA pellet was dried at the room

temperature. The dehydrated DNA pellet was re-suspended in 50 µl of PCR water. Measurements

of quality and quantity of extracted DNA were carried out by Nanodrop. DNA purification was

done to obtain effective and pure, good quality DNA.

34

4.3 PCR USING Vf PRIMERS

After the DNA extraction multiplex PCR reaction was carried out using pairs of Vf primers

(primers AL07-For, AL07-Rev, AM19-For, AM19-Rev) (Tartarini et al., 1999). Al-07 is a

codominant primer, while AM-19 is a dominant primer and both are specific primers for the Vf

gene. Primers AL07-For and AL07-Rev amplify 724 bp fragment linked to the susceptible allele

of the Vf gene and primers AM19-For and AM19-Rev amplify 526 bp fragment coupled with the

resistant allele of the Vf gene.

PCR amplifications (Table 3) were done in 20 µl volume containing 1 x Dream taq green master

mix, 0.2 µM of AL07-For and AL07-Rev, 0.1 µM AM19-For and AM19-Rev and ̴ 14 ng of

DNA.

Table 3. The program of amplification markers of resistance alleles of Vf gene in apple seedlings.

Steps Temperature Time Processes

1 94°C 120 s Initial denaturation

2 94°C 30 s Denaturation

3 60°C 60 s Annealing

4 72°C 120 s Elongation

5 Go to step 2 34x

6 72°C 600 s Elongation

7 4°C 24 h

DNA samples were further analyzed using agarose gel electrophoresis. Agarose gel (1.5%) was

prepared: to 1.05 g of agarose was added 70 ml TAE-buffer, after cooling for visualization of the

DNA, 35 µl of ethidium bromide (1 mg/ml) was added into the gels. Gel electrophoresis was run

at 80 volts for about 45 min and a picture from gel was taken.

35

4.4 CLASSIFICATION OF APPLE SEEDLINGS

In the autumn, apple seedlings were evaluated and categorized into three groups based on their

phenotypic characteristics: columnar type, potential columnar type and normal type. Columnar

type plants showed clear columnar phenotype with straight main stem and short internodes.

Potential columnar type plants showed some characteristics typical for columnar plants but could

not be categorized into this class with sufficient certainty. Normal type plants showed normal

growth habit with longer internodes and lateral branches.

36

5. RESULTS

5.1 DNA EXTRACTION FROM FRESH LEAVES

The extracted DNA concentrations are presented in Table 4. The highest DNA concentration was

1577.98 ng/µl, while the lowest 3.91 ng/µl. The mean value of DNA concentration was recorded

as 171.28 ng/µl. The purity of DNA was checked by measuring the absorbance ratio at A260/A280.

The highest A260/A280 ratio of DNA samples was 2.59, whereas the lowest was 1.07. Most of

samples had A260/A280 ratio approximately 1.75. A260/A230 ratio was used as a secondary measure

of DNA purity. The highest A260/A230 ratio was 2.64, while 0.41 was the lowest. The mean values

of A260/A230 ratio showed 1.55.

Table 4. The DNA concentration of analyzed samples taken from fresh apple leaves. n=103

5.2 PCR ANALYSIS TO DETECT Vf GENE

One hundred and three samples were successfully analyzed with respect to presence of PCR

products specific for Vf genes. Eight samples did not yield proper PCR products, thus these

samples were analyzed again. Out of all the analyzed samples only five samples did not yield any

PCR products. In total, resistant allele of Vf gene was detected from four seedlings (Figure 5).

DNA concentration

(ng/µl) A260/A280 ratio A260/A230 ratio

Highest 1577.98 2.59 2.64

Lowest 3.91 1.07 0.41

Mean 171.28 1.75 1.55

37

Figure 5. PCR products separated by agarose gel electrophoresis for detection Vf allele in samples

obtained from apple seedlings. ‘M’ – molecular marker (O’GeneRuler 100 bp DNA Ladder

(Thermo Scientific)), ‘1-111’ - numbers of samples, ‘+’positive control, ‘-‘ negative control. 526

bp fragment coupling with resistant allele of Vf gene, 724 bp coupling with susceptible allele.

38

5.3 CLASSIFICATION OF APPLE SEEDLINGS

In autumn, apple seedlings were categorized into three groups based on their growth habit:

columnar type, potential columnar type and normal type. Classification was done as described in

materials and methods. In total seventy-four seedlings were classified as columnar type growth

seedlings; thirty-eight of them were analyzed by PCR (Table 5).

Table 5. Classification of apple seedlings. PCR = Plants were analyzed by PCR to detect resistant

allele of Vf gene. NA = Not analyzed by PCR.

Classification of Apple Seedlings

Columnar type Potential columnar type Normal type Indoor garden

PCR NA PCR NA PCR NA PCR

Plants 38 36 26 45 23 43 16

Vf gene 2 - 1 - - - 1

According to the results of PCR four seedlings have resistant Vf gene. Two of them were

identified to have apple scab resistance Vf gene and classified as columnar apple seedlings. From

twenty-six analyzed samples classified into group of potential columnar type seedlings one was

detected to have Vf gene. One sample carrying resistant Vf gene was detected from indoor garden

seedling.

39

6. DISCUSSION

The purpose of this research work was to identify the resistant allele of Vf gene, which confers

resistance against apple scab disease, in columnar phenotype apple seedlings by PCR. Leaf

samples for DNA extraction were collected from the apple seedlings with the potential of being

columnar type and DNA extraction was done using the CTAB method followed by the multiplex

PCR reaction. Two plants were confirmed to have Vf gene in combination with columnar growth

type and one plant having Vf gene, which was classified into group of potential columnar

phenotype. Additionally, it was detected that one sample taken from seedling from indoor garden

also has Vf gene, but without columnar phenotype.

The molecular markers used in this study, were SCAR markers that are based on PCR and

specific primers. The PCR method used for the identification of Vf gene worked well. The results

of PCR amplifications were reliable over a wide range of DNA concentrations as from all

analyzed samples 103 samples were successfully analyzed and only five samples failed. The

major advantages of SCAR markers are that they are fast, less sensitive to reaction conditions,

easy to use, reliable and productive (Abdin et al., 2012). As recently reviewed by Kiran et al.

(2010), these markers represent single locus, which is detected by amplification of genomic DNA

with specific primers, and normally result in reproducible and sharp band. Due to the low cost

and high reproducibility, SCAR markers are proven a valuable tool and practical way for

screening numerous samples at once (Li et al., 2012; Kiran et al., 2010).

The good quality of the DNA is the prerequisite for successful PCR analysis. The DNA

extraction method used in this study, based on the CTAB, is well established and widely used

with plant samples. The method developed by Doyle and Doyle (1987) worked successfully with

fresh apple leaves and yielded satisfactory results for the further PCR amplification. The quality

of extracted DNA was quite good: the absorbance ratios of most analyzed samples were

acceptable. Besides, in most of the samples, the DNA quality was good enough for PCR, for the

reason that there were only five samples that did not produce any amplification products. The

method applied in this study is also inexpensive. On the other hand, DNA extraction with this

method is time-consuming (Chabi Sika et al., 2015; Bellstedt et al., 2010).

40

Bellstedt et al. (2010) have developed a rapid DNA extraction protocol that was proved to be

suitable for various plant species. Compared to CTAB method, it reduces the time of DNA

extraction procedure from 24-48 hours to almost 2 hours. Moreover, the method is cost-effective

as well as easy to perform, thus excellent for large sample numbers from any plant materials.

Consequently, the method recommended by Bellstedt et al. (2010) could be appropriate for apple

as well, and it might be worthwhile to test it together with Vf gene and PCR to make the selection

process of apple seedlings even more efficient.

During this research work, one of the key steps was the identification of columnar apple

seedlings. As stated by Otto et al. (2013), the clear detection of columnar apples is difficult at the

early stage of growth, because of the presence of many growth phenotypes and various

intermediary types. This was also the case in this study: seventy-one plants could not be clearly

classified either as columnar type or normal type plants, but were categorized to potential

columnar type (Table 5). In addition, twenty-three plants that were identified as columnar type at

the sampling stage in summer were categorized as normal growth type in autumn when the

seedlings were evaluated again.

To improve the reliability of the identification of columnar seedlings, molecular markers could be

exploited. Tian et al. (2005) have found several DNA based molecular markers linked to Co

gene. Studies found out that SCAR and SSR markers are appropriate due to their ease to apply

and steadiness. Bendokas et al. (2007) reported the usage of molecular markers for the

identification of columnar type apple seedlings at the juvenile stage. Usage of SCAR markers in

the identification of columnar growth type seedlings is helpful as it makes the selection of

columnar seedlings faster therefore reducing the time of breeding (Bendokas et al., 2007).

According to results obtained in this study, it can be stated that PCR process is a useful way to

identify apple scab resistance Vf gene from apple seedlings in juvenile phase. PCR method is

itself considered to be useful in many other fields of breeding traits.

41

7. CONCLUSIONS

In this study, the results confirm that Vf genes were combined columnar growth type and the PCR

markers that were used are useful in the detection of the apple scab resistance among seedlings.

The selection of apple scab resistance seedlings can be facilitated by using molecular markers

closely linked to resistance genes in the early seedling stage. Molecular markers associated

selection and breeding, for main phenotypic features, can be a significant tool that reduces

duration and costs of breeding programs.

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