1

Chapter 1

Apples

J. F. Hancock 1, J. J. Luby 2, S. K. Brown 3, G. A. Lobos 4

1
Department of Horticulture, 342C Plant and Soil Sciences Building, Michigan State
University, East Lansing, Michigan 48824, U.S.A. 2 Department of Horticultural Sciences,
329 Alderman Hall, University of Minnesota, St Paul, Minnesota, 55413, U.S.A. 3
Department of Horticultural Sciences, 630 W. North Street, Cornell University, Geneva, New
York 14456, U.S.A. 4 Laboratorio de Ecofisiologia Vegetal, Escuela de Agronomia,
Universidad de Talca, Chile
E-mail: [email protected]

Abstract

The overall objectives of modern apple breeding programs are to increase the marketability of fruit
and reduce production costs. Developing well adapted cultivars with resistance to major pests is also
a focus of all breeding programs. The apple is generally grown as a composite tree with a rootstock
and a fruiting scion, making rootstock breeding as important as the development of scion cultivars.
Genetic resistance has been found for a number of the major pests of apple. Engineering resistance
to apple scab and fire blight has been the focus of a number of laboratories. Most of the traits
associated with adaptation and productivity have been shown to be quantitatively controlled, including
chilling requirement, cold hardiness, plant vigor, season of flowering and duration of the juvenile
period. Many of the traits associated with fruit quality are also quantitatively inherited including flavor,
skin color, shape, size and texture. Several cDNA libraries have been developed to identify genes
associated with pollination and apple fruit development. A number of apple linkage maps have been
published using several different sets of parents and molecular markers have been linked to a
number of monogenic traits. Mining of existing apple EST information promises to expand our
knowledge of many genes important in the genetic improvement of apple.

1.1 Introduction

Apples are cultivated all across the temperate world. Their adaptive range extends from the
extreme cold of places such as Siberia and North China to the much warmer environs of
Columbia and Indonesia. More than 60 countries produce over 1000 or more metric tons of
apples, with China, U.S.A., Turkey, Iran, France, Italy, Poland and Russia being the leading
producers. World production now exceeds 57,000 million metric tons (FAOSTAT, 2004).

Apples are an extremely versatile crop. They can be eaten directly from the tree or stored for
up to a year in controlled atmospheres. They can be processed into juice, sauce and slices,
and are a favorite ingredient in cakes, pies and pastries. The juice can be consumed fresh or
fermented into cider, wine or vinegar. The ornamental crab apples are also known for their
floral display and attractive foliage.

There are over 6,000 regionally important cultivars and land races across the world, but a few
major cultivars now dominate world fruit production (O’Rourke 2003). 'Delicious' is the most
important cultivar grown, followed by 'Golden Delicious', 'Granny Smith', 'Fuji' and 'Gala'.
These varieties represent over 60% of the world’s production. Emerging varieties include
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'Cripps Pink' (often sold under the trademark Pink Lady®), 'Honeycrisp' (sold in Europe as
Honeycrunch®) (Figure 1.1), 'Scifresh' (fruit marketed under the trademark Jazz®),
'Delblush' (fruit sold as Tentation®), 'Civni' (fruit marketed as Rubens®, 'Corail' (fruit
marketed as Pinova® or Pinata®) and 'Ariane'.

Figure 1.1. Fruit of the Honeycrisp apple cultivar (photographed at the University of
Minnesota Horticultural Research Center, Excelsior, Minnesota, U.S.A.).

The genetic base of the cultivated apple has greatly eroded over time as regional cultivars
have been replaced. This has been compounded by the loss of many public apple breeding
projects and their associated apple cultivar collections (Brooks and Vest 1985). Forsline and
his group at the USDA Germplasm Repository at Cornell University has worked hard to
counter this trend by actively collecting and cataloging native apple germplasm and making it
available to apple breeders (Forsline et al. 1994, Hokanson et al. 1997).

1.2 Evolutionary biology and germplasm resources

The genus of apples, Malus, belongs to the subfamily Pomoideae of the Rosaceae family.
Another important fruit tree, pear (Pyrus), belongs to the same subfamily. There are over 30
primary species of apple and most can be readily hybridized (Korban 1986, Way et al. 1991).
The cultivated apple is likely the result of initial domestication followed by inter-specific
hybridization (Harris et al. 2002). Its primary wild ancestor is M. sieversii whose range is
centered at the border between western China and the former Soviet Union. Apples are the
main forest tree there and display the full range of colors, forms and tastes found in

domesticated apple has been referred to with the epithet Malus ×domestica (Korban and
domesticated apples across the world (Forsline et al. 1994, Hokanson et al. 1997). The

Skirvin 1984), although recently Mabberley et al. (2001) proposed that Malus pumila should
properly refer to the domesticated apple and its presumed wild relative M. sieversii. Other
species of Malus which contributed to the genetic background of the apple likely include: M.
orientalis of Caucasia, M. sylvestris from Europe, M. baccata from Siberia, M. mandshurica
from Manchuria, and M. prunifolia from China. It is likely that these species hybridized with
domesticated apples as they were spread by humans (Harris et al. 2002).

The bulk of the apple species are 2n = 2x = 34 (Table 1.1), although higher somatic numbers
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of 51, 68 and 85 exist; several of the cultivated types are triploid (Chyi and Weeden 1984). It
is possible that the high chromosome number of apple represents an ancient genomic
duplication, since there are several other Rosaceous fruit species with lower haploid
chromosome numbers of n = 8 and 9. Based on cytology and analysis of morphological
characters, the Maloideae likely have a polyploid origin (Phipps et al. 1991). Isozyme studies
in Malus support an allopolyploid origin based on the presence of duplicated gene systems,
allele segregation and fixed heterozygosities (Chevreau et al. 1985, Weeden and Lamb 1987,
Dickson et al. 1991). An allotetraploid origin involving ancestral Spiroideae (mostly x = 9)
and Amygdaloideae (x = 7) was proposed by Sax (1931 and 1933) and is supported by
flavonoid chemistry (Challice 1974, Challice and Kovanda 1981) and morphological traits
(Phipps et al. 1991). Apples are largely self-incompatible and some are apomictic. They are
propagated vegetatively, usually as composites with a separate rootstock and scion.

Table 1.1. Distribution of selected apple species in subsection Pumilae and their chromosome numbers (adapted
from Way et al. 1991).

Chromosome
Species Distribution
number (2n)
M. asiatica Nakai 34 N. & N.E. China, Korea

M. baccata (L.) Borkh. 34, 68 N. & N.E. China

M. ×domestica 34, 51, 68 Worldwide

M. floribunda (Siebold) ex. Van Houtte 34 Japan

M. halliana Koehne 34 Japan

M. hupehensis (Pamp.) Rehder 51 Central China

M. mandshurica (Maxim.) Kom. ex Skvortsov 34 Manschuria

M. micromalus Makino 34 S.E. China, Korea

M. orientalis Uglitzk. ? Caucasia

M. prunifolia (Willd.) Borkh. 34 N. & N.E. China, Korea

M. pumila Mill. 34 Europe

M. sieversii (Ledeb.) M. Roem. ? N.W. China

M. spectabilis (Aiton) Borkh. 34, 68 China

M. sikkimensis (Wenz.) Koehne ex C. K. Schneid. 51 Himalaya

M. sylvestris (L.) Mill. 34 Europe

Apples were certainly one of the earliest fruits to be gathered by people, and their
domestication was probably preceded by a long period of unintentional planting via garbage
disposal. It is difficult to determine exactly when the apple was first domesticated, but the
Greeks and Romans were growing apples at least 2,500 years ago. They actively selected
superior seedlings and were budding and grafting 2,000 years ago (Janick et al. 1996). People
in Central Asia where M. sieversii is native still save desirable trees when the forest is cleared
for agriculture (Ponomarenko 1983) and commonly graft and plant desirable M. sieversii
from the forest into their gardens. Planting desirable trees from root suckers may also have
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been a common practice prior to grafting, as M. sieversii trees sucker freely. Conversely,
people may have cloned and moved some of their horticulturally desirable trees to areas
where they seasonally grazed their animals. These trees or their open pollinated descendants
may be among the horticulturally elite specimens observed in some of the forests today.

The most likely beginning of cultivation was in the region between the Caspian and Black
seas (Vavilov 1949-1950); apple cultivation had reached the Near East by 3,000 B. P.
(Zohary and Hopf 1993). The Romans spread the apple across Europe during their invasions
and it was dispersed to the New World by European settlers during the sixteenth century.

The passage of trade routes from China to the Middle East and Europe through Central Asia

intentionally or unintentionally, of M. sieversii and its hybrid derivatives. The M. ×domestica

probably facilitated repeated short and long distance dispersal to the east and west, either

Borkh complex may have arisen through hybridization with species native to China including
M. prunifolia (Willd.) Borkh., M. baccata (L.) Borkh., M. mandshurica (Maxim.) Kom. ex
Skvortsov, and M. sieboldii (Regel) Rehder. To the west, hybridization with the local species
M. sylvestris (L) Mill. and M. orientalis Uglitzk. is conjectured (Ponomarenko 1983, Morgan
and Richards 1993, Hokanson et al. 1997, Juniper et al. 1999).

During the late nineteenth and twentieth centuries, M. ×domestica cultivars found or bred in
Europe, Russia, North America, New Zealand, Japan, and Australia were introduced

et al. 1991, Janick et al. 1996). Several species are known to have contributed to the M. ×
throughout the world and form the basis for most current commercial apple production (Way

Houtte, M. ×micromalus Makino, M. ×atrosanguinea (hort ex Späth) C.K. Schneid., M.

domestica complex in modern breeding programs including M. floribunda Siebold ex Van

baccata (L.) Borkh., M. zumi (Matsum.) Rehder, and M. sargentii Rehder (Ponomarenko
1983, Way et al. 1991, Janick et al. 1996).

In southern and eastern Asia, Nai, or the Chinese soft apple, M. ×asiatica Nakai, was the
primary cultivated apple in China and surrounding areas for over 2000 years until M. ×

Richards 1993, Zhang et al. 1993, Watkins 1995, Zhou 1999). Malus ×asiatica is likely a
domestica was introduced in the late nineteenth and early twentieth centuries (Morgan and

hybrid complex derived primarily from M. sieversii with M. prunifolia and perhaps other
species.

Prehistoric remains and historical records, reviewed by Morgan and Richards (1993), provide
evidence of the cultivation, dispersal, and human use of the apple in the Asia and Europe
over the last several thousand years. Archaeological remains of apple that dated to about
6500 BC were found in Anatolia, though it is impossible to know the source of this fruit or
whether it was cultivated. Historical evidence referring to apple cultivation dates to the
second millennium BC from Anatolia, and northern Mesopotamia. By 500 BC, the apple
likely was cultivated widely throughout the Persian Empire as fruit orchards are prominently
featured in writings from the period. When Alexander the Great conquered the Persians
around 300 BC, the cultivation of fruits was dispersed throughout the Greek world. By this
time, the Greek philosopher, Theophrastus, distinguished the sweet cultivated apple from
astringent wild forms.

The ascendance of the Roman Empire spread cultivation of the domesticated apple north and
west through Europe where it supplanted and likely hybridized with, the native crab apple,
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M. sylvestris. Multiple varieties were recorded by the Roman writer, Pliny, and they attained
an important place in Roman cuisine, medicine, and aesthetics by the first century AD. The
Roman goddess Pomona was revered as the deity associated with apple and other fruits. With
the rise and spread of Christianity and Islam over the next several centuries, apples were
carefully maintained, even through wars and difficult times, in the abbey gardens throughout
Europe and the orchards of Iberia. These apparently replaced the native crab apples that had a
place in the diet of early Celts, Gauls, Franks, Scandinavians and other peoples of northern

as a basic monastic skill and many abbeys developed large orchards with many M. ×
Europe in fermented, dried, or cooked forms. Maintenance of fruit gardens was encouraged

domestica cultivars. Likewise in the Muslim world of the eastern Mediterranean and Iberia,
fruit growing was revered in keeping with Koranic teachings and skills of grafting, training
and pruning became highly developed.

Today, the largest collection of apple germplasm is held at the Plant Genetics Resource Unit
at Cornell University, Ithaca New York, where there are almost 4,000 accessions being
maintained (http://www.ars.usda.gov/main/site_main.htm?modecode=19100500). Many of
these genotypes were collected from the apples center of diversity in Central Asia (Hokanson
et al. 1997, Forsline 2003).

1.3 History of improvement

From the thirteenth century, apples became more and more widely planted throughout Europe
in gardens of royalty and commoners. Raw apples were occasionally consumed, but they
were more greatly prized when cooked and sometimes blended with spices and sugar or
honey. Fermented juice, or cider, like beer, was preferred to the sometimes questionable local
water supply. By the seventeenth century there were at least 120 cultivars described in
western Europe. The rise and spread of Protestantism, which saw the apple as the special fruit
of God, is credited with expanding apple cultivation across northern and eastern Europe after
beginning in Germany in the early seventeenth century. By the end of the eighteenth century,
many hundreds of cultivars were recognized throughout Europe. The Royal Horticultural
Society of England acknowledged at least 1200 in 1826. The eighteenth and nineteenth
centuries saw apple cultivars recognized and classified based on their suitability for their end
uses. Aromatic dessert apples were more widely appreciated by this time, while good cooking
types were still appreciated for puddings and pastries. Flavorful varieties with moderate acid
and tannin levels were prized for cider production. The late nineteenth and early twentieth
centuries represented the maximum of diversity in apple cultivation in Europe with hundreds
of locally popular varieties being grown in thousands of small orchards. In the twentieth
century, the rise of imported fruit from the Americas, New Zealand, Australia, and South
Africa forced European orchards to increase in size and decrease in number and, to a large
extent, to adopt the very same cultivars that were developed in, and imported from the New
World.

Apples were established in the 1650s near Cape Town in South Africa to sustain settlers and
to supply the ships of the Dutch East India Company. The commercial orchard district in the
Western Cape apple was started by Cecil Rhodes and his associates in the late nineteenth and
early twentieth century to replace a faltering wine industry.

Apples were introduced to Australia, on the island of Tasmania and at the present site of
Sydney, in 1788. Orchards were established by settlers in Tasmania and New South Wales by
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the early 1800s. Significant production areas were eventually developed in Tasmania and the
southeastern mainland. In 1814, English missionaries brought apples from Australia to New
Zealand where two large apple production districts became established in the districts of
Hawkes Bay and Nelson during the nineteenth and twentieth centuries.

Beginning in the sixteenth and seventeenth centuries, European colonists brought apples to
the Americas. Spanish priests introduced them to their missions in Chile and California.
Spanish and Portuguese settlers introduced apples to their settlements in suitable temperate
climate zones of South America. European settlers brought apple seeds to establish orchards
in the eastern United States and Canada. Apples grew well from northern Georgia through
eastern Canada and, as in Europe, were soon highly prized for food and drink, and as a source
of sugar and alcohol. The first orchards in New England were recorded in the 1620s and
1630s and became important components of the New England farmstead. Likewise, they
became important on the large plantations of the mid-Atlantic colonies by the mid-1700s,
including those of the early United States presidents, George Washington and Thomas
Jefferson. Jefferson, an astute horticulturist, acquired and carefully trialed dozens of cultivars
for his Monticello gardens in Virginia.

In Canada, French colonists established orchards in the seventeenth century along the St.
Lawrence Valley. Settlers also established orchards around Lake Ontario, and in the milder
valleys of Nova Scotia and New Brunswick.

As settlers moved westward in the United States, apple orchards were a requirement of
homesteading throughout the territories of the Ohio River Valley. Jonathan Chapman, known
as Johnny Appleseed, devoted his later life, from 1806 to 1847, to helping settlers establish
thousands of apple trees on their new farms in the Ohio River drainage. The Great Lakes
region of the United States, especially the states of New York, Michigan, and Ohio, continues
to be a major apple production area.

In 1847, as settlers moved in to the productive valleys of western Oregon, Washington and
northern California, Henderson Llewelling brought 700 trees with his family on the Oregon
Trail and eventually established the first fruit nursery in the Pacific Northwest. As irrigation
schemes were eventually developed the Pacific Northwest, especially including the basin of
the Columbia River and its tributaries west of the Cascade Mountains and extending to the
Okanagon River valley in British Columbia, eventually became one of the preeminent apple
production areas of the world.

By the early twentieth century, the United States and Canada were the two largest apple
producing nations. Later in the century the Soviet Union also became important. By the
beginning of the twenty-first century China has become the largest apple producer, with a
large proportion of the crop being exported as concentrated juice. Major southern hemisphere
production, much of it for export to northern hemisphere countries during their spring and
summer, occurs in South Africa, Chile, Argentina, New Zealand, and Australia. As
previously mentioned, production is currently dominated by strains of just a few cultivars:
'Delicious', 'Golden Delicious', 'McIntosh', and 'Jonagold' from North America; 'Braeburn'
and 'Gala' from New Zealand; 'Granny Smith' from Australia; and 'Fuji' from Japan. Though
many other cultivars remain locally important, these dominate current production and are
also widely used in breeding programs around the world.
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From its origins among the millions of wild M. sieversii trees in the mountains of central Asia
(Figure 1.2), and from the early development of thousands of local cultivars in Europe and
America, the domesticated apple, as cultivated in twenty-first century, has shrunk drastically
in diversity.

Figure 1.2. Photo showing the diversity of fruit collected from wild Malus sieversii apple
trees in the Tarbagatai mountains of eastern Kazakhstan.

1.4 Current breeding efforts

The overall objectives of modern breeding programs are to increase the marketability of fruit
and reduce production costs. Apples are sold fresh, juiced and processed in numerous ways,
but the largest overall market involves fresh fruit. Numerous apple species are also important
as ornamentals (Fiala 1994).

Dessert apples are sold primarily based on appearance (size, color, shape and freedom from
blemishes) and quality (taste and texture) (Janick et al. 1996; Laurens 1999; Brown and
Maloney 2003). There is considerable regional variation in taste preferences, from a desire
for tartness in Europe and the U.S. Midwest, to a preference for sweetness and low acidity in
Asia. Low allergenic apples have become a priority in Europe. Favored colors range widely
from solid green, yellow to red and bicolors of many combinations. In general, apples are
expected to be blemish free, large (> 70 mm in diameter), and ovate or conic shaped. Storage
life is also a critical parameter, as most apples are stored for long periods of time. Resistance
to apple scab and powdery mildew are common breeding goals. Niche markets are also
arising for improved nutritional aspects such as higher antioxidants.

The attributes needed for processed fruit depends on their final market. Some of the most
important markets are for cider, sauce and slices (Crassweller and Green 2003). Less
browning is a particularly critical parameter in the fresh cut and slices market.

Production costs are greatly reduced by maximizing yields, increasing picking efficiency and
incorporating disease and pest resistance. Adaptation is a key parameter associated with
yield, particularly in marginal climates with extreme winters or low chilling hours. Pest and
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disease resistance is critical to productivity in areas where other means of control are not
available or undesirable. This applies particularly to growers interested in producing
“organic” apples.

The apple is generally grown as a composite tree with a rootstock and a fruiting scion,
making rootstock breeding as important as the development of scion cultivars. There are a
number of important attributes of rootstocks including; ease of propagation, clean upright
stems, easy to bud or graft, well anchored root systems, no suckering, and good stock-scion
compatibility (Janick et al. 1996). Rootstocks should also offer a range of tree size control
from dwarfing to vigorous, induce early, heavy cropping, tolerance to cold and wet or dry
soils, and be resistant the prevailing pests and diseases (Webster and Wertheim 2003).

Brown and Maloney (2003) reviewed current breeding programs and activities throughout
the world. In the US, a new program was started in 1994 at Washington State University.
Other programs in the US include the PRI (Purdue University, Rutgers University and the
University of Illinois) cooperative that has concentrated on developing scab resistant
cultivars, the University of Minnesota (of 'Honeycrisp' fame), and Cornell University, best
known for 'Empire' and 'Jonagold'. The New Zealand program has been an innovator in the
licensing and restricted availability of selections from their program. Increasingly, programs
are partnering with private industry, examples include the collaboration between breeders,
nurseries and packers in France (Lauren et al. 2004).

1.5 Genetics of economically important traits

1.5.1 Pest and disease resistance

Genetic resistance has been found for a number of the major pests and diseases of apple
including: Fire blight (Erwinia amylovora), Alternaria blotch (Alternaria mali), apple blotch
(Phyllosticta solitaria), apple canker (Nectria galligena), apple scab (Venturia inaequalis),
cedar apple rust (Gymnosporangium juniperi-virginianae), crown rot (Phytophthora
cactorum), powdery mildew (Podosphaera leucotricha), wooly apple aphid (Eriosoma
lanigerum), rosy apple aphid (Dysaphis plantaginea) and rosy curling aphid (D. devecta)
(Table 1.2).

Resistance to Alternaria blotch, crown rot, wooly apple aphid, rosy apple aphid and rosy
curling aphid are regulated by a single dominant gene. RFLP markers have been found for
resistance to rosy leaf curling aphid (Roche et al. 1997). Apple blotch and apple rust
resistance are regulated by two dominant genes, and a number of polygenes. Genes for
resistance to powdery mildew have been identified and markers have been developed: Pl1
(Markussen et al. 1995), Pl2 (Dunemann et al. 1999), Pl-d (James et al. 2004), Pl-w (Evans
and James 2003). A marker has yet to be developed for Pmis from Mildew immune seedling
(MIS). Resistant Malus genotypes have also been identified for fire blight and apple canker,
although the genetics have not been elucidated. 'Delicious' has fairly good resistance to fire
blight, but immunity is only found outside of the cultivated species of apple (Janick et al.
1996).
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Table 1.2. Genetics of pest and disease resistance in apple.

Pest or disease Observations and source

Bacterial
Fire blight Immunity is present in some Malus species (Janick et al. 1996); QTLs for
Erwinia amylovora resistance reported by Khan et al. (2006)

Fungi
Alternaria blotch Resistance is controlled by a single dominant gene (Ralt) which is epistatic to
Alternaria mali a dominant gene (Alt) controlling susceptibility (Saito and Niizeki 1988)
Apple blotch Susceptibility is regulated by two dominant genes (Ps1 and Ps2) with
Phyllosticta solitaria duplicate recessive epistatic interaction between gene pairs (Mowry and
Dayton 1964)
Apple canker Highly resistant cider apples and rootstocks have been identified (Moore
Nectria galligena 1960)
Apple scab Both quantitative and qualitative resistance exists; major source is a
Venturia inaequalis dominant gene, Vf ; multiple resistance genes at the Vf locus have been found
in several species; the resistance of Vf is enhanced by polygenes (Dayton
and Williams 1968, Crosby et al. 1992, Bus et al. 2002); numerous QTL and
markers identified (Tartarini and Sansavini 2003, Durel et al. 2004, Bus et
al. 2005a and b, Hemmat et al. 2003)
Cedar apple rust Regulated by two, dominant genes (Gy-a and Gy-b) and perhaps other
Gymnosporangium modifying genes; resistance mechanisms vary (Mowry 1964, Aldwinckle et
juniperi-virginianae al. 1977, Chen and Korban 1987)
Crown rot Regulated by a single dominant gene (Pc), but polygenes are important
Phytophthora cactorum (Alston 1970b, Watkins and Werts 1971)
Powdery mildew Regulated by several dominant genes (Pl1 and Pl2) and polygenes; resistance
Podosphaera leucotricha may be enhanced by polygenes (Alston 1977, Korban and Dayton 1983,
Gallott et al. 1985); quantitative resistance will be needed for durable
resistance (Caffier and Parisi 2007); markers identified for the dominant
alleles Pl1, Pl2, Pl-d, Pl-w, Pl-m (Markussen et al. 1995, Durel et al. 2002,
Evans and James 2003)

Insects
Wooly apple aphid Regulated by a single dominant gene (Er); 'Northern Spy' has high level of
Eriosoma lanigerum resistance, along with several other cultivars; resistance gene is closely
linked to incompatibility gene (Knight et al. 1962, Knight 1972, Cummins et
al. 1981); markers identified for Er1 and Er3 (Sandanayaka et al. 2003)
Rosy apple aphid Regulated by a single dominant gene (Smh); resistance found in open
Dysaphis plantaginea pollinated selection of M. robusta (Alston and Briggs 1970)
Rosy curling aphid Regulated by a single dominant gene; four different resistance genes have
D. devecta been identified in 'McIntosh' (SDpr) 'Cox’s Orange Pippin' (Sd1), 'Northern
Spy' (Sd2) and Malus robusta (Sd3); a precursor gene must be present for
effective resistance (Alston and Briggs 1977); markers identified to
Sd1(Roche et al. 1997, Cevik and King 2000)

At least 10 different resistance genes have been identified for apple scab (Bus et al. 2002),
and one of them, Vf from the ornamental crabapple M. floribunda 821, has been used all over
the world to create new scab resistant cultivars (Laurens 1999). This gene has been cloned
and shown to confer scab resistance to a transgenic cultivated variety 'Gala' (Belfanti et al.
2004). Scar markers have been identified for Vbj from Malus baccata jackii (Gygax et al.
2004) and Vm from M. atrosanguinea 804 (Cheng et al. 1998). Patocchi et al. (2005) used
genome scanning to identify a microsatellite tightly linked to Vm. Numerous other QTL have
been identified for scab resistance, which will be described in the section on genetic mapping
of apple. Gessler et al. (2006) review scab resistance in apple.
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In breeding for multiple disease or pest resistance a balance of resistance and commercial
fruit quality may be difficult to achieve. Adequate levels of resistance to the major fungal
pathogens (scab and powdery mildew) coupled with resistance to fire blight requires large
populations and great attention to other secondary problems such as leaf spot, moldy core and
summer fruit pathogens.

Attempts have been made to associate specific enzyme activities with resistance to superficial
storage scald. The total activities of guanacol-dependent peroxidase (POX),
superoxidismutase (SOD) and catalase (CAT) were not found to be significantly associated
with susceptibility to scald in a segregating population of 'White Angel' x 'Rome Beauty';
however, there were associations with the presence and absence of individual isozymes
(Kochhar et al. 2003).

Several genes have been isolated that are related to disease resistance. A c-DNA has been
cloned from fruit of 'Fuji' that encodes a pathogenesis-related 5/thaumatin-like protein
(PR5/TL) that was named Mdt 1 (Malus domestica thaumatin-like protein) (Oh et al. 2000).
A salicylate-inducible PR-10 gene (designated as APa) was found to be expressed during
infection of a compatible vs. a non-compatible race of Venturia inaequalis (Poupard et al.
2003). Eighteen genes were identified as having higher expression levels during infection of
'Golden Delicious' by Penicillium expansum (Sánchez-Torres and González-Candelas 2003).
Two of these genes likely encoded ß-glucosidase and phosphatase 2C.

A number of apple sequences have been identified that are similar to the R (resistance) genes
of other plants that contain a nucleotide binding site (NBS). NBS-containing genes are the
most common class of resistance genes found in plants. Over 20 families of NBS-containing
genes have been identified in apple that include the two major groups described in dicot
plants, one lacking a toll-interleukin element and one containing it (Baldi et al. 2004, Calenge
et al. 2005). A cluster of receptor-like genes has been identified in bacterial artificial clones
derived from the Vf scab resistance locus that are similar to the Cladosporium fulvum (Cf)
resistance gene family of tomato (Vinatzer et al. 2001).

1.5.2 Morphological and physiological traits

Most of the traits associated with adaptation and productivity have been shown to be
quantitatively controlled, including chilling requirement, cold hardiness, plant vigor, season
of flowering and duration of the juvenile period (Table 1.3).

Several aspects of plant habit have been shown to be regulated by single genes in inheritance
studies (Alston et al. 2000). A dominant gene regulates the columnar habit in the 'Wijcik'
clone of 'McIntosh' (Lapins and Watkins 1973, Lapins 1974). A series of recessive alleles
have been identified that regulate dwarfing (Decourtye 1967, Alston 1976). Recessive genes
have been associated with the spur-habit in sports of 'Golden Delicious', 'Redspur' and
'Starkrimson' (Decourtye and Lantin 1969, Alston and Watkins 1973). RAPD markers have
been identified for terminal bearing, initial bud break, root sucker formation (Weeden et al.
1994) and columnar tree habit (Hemmat et al. 1997).
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Table 1.3. Genetics of adaptation, productivity, plant habit and fruit quality in apple.

Attribute Observations and source

Adaptation
Chilling requirement Generally quantitatively inherited, although the low chill requirement of
'Anna' is thought to be controlled by one major gene and a number of minor
ones (Hauagge and Cummins 1991, Labuschagne et al. 2002); bud break
number is highly heritable (Labuschagne et al. 2003)
Cold hardiness Quantitatively inherited, largely additive (Watkins and Spangelo 1970)
Growth rate Quantitatively inherited; QTL identified (Conner et al. 1998)
Vigor Quantitatively inherited, largely additive (Watkins and Spangelo 1970, Durel
et al. 1998)
Season of flowering Quantitatively inherited, largely additive (Janick et al. 1996); QTL identified
(Liebhard et al. 2003a)
Harvest date Quantitatively inherited; QTL identified (Liebhard et al. 2003a)
Productivity
Flower number Quantitatively inherited; QTL identified (Liebhard et al. 2003a)
Fruit number Quantitatively inherited; QTL identified (Liebhard et al. 2003a)
Juvenile phase length Quantitatively inherited; QTL identified (Liebhard et al. 2003a)
Incompatibility Numerous S-alleles exist, are semi-compatible combinations (Broothaerts et
al. 2004)
Plant habit
Compact Single dominant gene (Co) identified for compact columnar habit in 'Wijcik'
clone of 'McIntosh'; genetics more complex in other sources of the compact
branching habit (Lapins and Watkins 1973, Lapins 1974); markers identified
(Conner et al. 1997, Kim et al. 2003, Tian et al. 2005)
Dwarfing Several sources of dwarfing exist that are regulated by recessive alleles (d1 –
d4) (Decourtye 1967, Alston 1976)
Internode length and Nº Quantitatively inherited; QTL identified (Conner et al. 1998)
Spur bearing habit Generally quantitatively inherited, but spur-habit in sports of 'Golden
Delicious', 'Redspur' and 'Starkrimson' result from a single recessive gene
(Decourtye and Lantin 1979, Alston and Watkins 1969)
Russett Single dominant gene identified with many modifying polygenes (Alston and
Watkins 1973, Durel et al. 1998); some russeted clones do not transmit russet
to offspring
Fruit quality
Acidity Quantitatively inherited; QTL identified for malic acid gene, Ma (Maliepaard
et al. 1998, Liebhard et al. 2003a)
Bioactive compounds High variability among cultivars in ascorbic acid and phenolic compounds
(Schmitz-Eiberger et al. 2003); QTLs identified by Davey et al. (2006)
Firmness Quantitatively inherited; QTL identified (Seymour et al. 2000, Liebhard et al.
2003a)
Skin color Quantitative but may be regulated by a few major genes; one possibility is
that three dominant, major genes produce color (Lespinasse et al. 1988,
Brown 1992); marker identified for a fruit color gene (Rf) regulating the
red/yellow dimorphism (Cheng et al. 1996); EST research indicated that myb
transcription factors are important (Espley et al. 2007, Takos et al. 2006)
Shape Quantitatively inherited with a low genotype by environment interaction
(Currie et al. 2000)
Size/weight Quantitatively inherited (Durel et al. 1998); highly heritable (Volz et al.
2001); QTL identified (Liebhard et al. 2003a)
Storage disorders High heritability for soft scald and superficial scald; moderate heritability for
water core; low heritability for external pit, internal pit, brown heart,
breakdown and chilling injury (Volz et al. 2001)
Sugar content Quantitatively inherited; QTL identified (Liebhard et al. 2003a)
Texture Quantitatively inherited (Durel et al. 1998); QTL identified (Seymour et al.
2002); Md-ACS1 found to be closely associated with fruit softening
 12

Table 1.4. MADS-box genes cloned and characterized in apple.

Gene Observations and source

AFL1 Expressed only in the floral bud during the transition from vegetative to reproductive growth.
Orthologue of Arabidopsis FL (Wada et al. 2002)
AFL2 Expressed in vegetative shoot apex, floral buds, floral organs and root. Orthologue of
Arabidopsis FL (Wada et al. 2002)
MdMADS1 Expressed in all floral organs and young fruits, but not in leaves. Expression highest in early
stages of flower and fruit development. Shows significant sequence homology with
Arabidopsis AGL2 (Sung and An 1997)
MdMADS2 Transcribed in all four floral organs and at all stages of flower development. Member of
SQUA subfamily of snapdragon. Transgenic tobacco expressing it flowered early and had
shorter bolts, but showed no homeotic changes in the floral organs (Sung et al. 1999)
MdMADS3 Expressed in the inner three whorls of the floral primordium, but not in fruit. Showed high
sequence homology with Arabidopsis AGL2 and AGL4 (Sung et al. 2000)
MdMADS4 Expressed ubiquitously in inflorescence meristem, floral meristem, fruit and seeds. Showed
high sequence homology with Arabidopsis AGL2 and AGL4 (Sung et al. 2000)
MdMADS5 More strongly expressed in fruit than flower buds; not expressed in leaves. Highest
expression in young fruit. Most strongly expressed in cortex and skin, little expression in
core. Showed high sequence homology with Arabidopsis AP1 (Yao et al. 1999). Expressed
specifically in sepals during flower bud formation (Kotoda et al. 2000). Transgenic
Arabidopsis with this gene flowered early (Kotoda et al. 2002)
MdMADS6 More strongly expressed in fruit than flower buds. Highest expression in young fruit. Most
strongly expressed in cortex and skin, but significant expression in core. Showed high
sequence homology with PrMADS1 and MdMADS7 (Yao et al. 1999)
MdMADS7 More strongly expressed in fruit than flower buds. Highest expression in older fruit. Most
strongly expressed in cortex and skin, but significant expression in core. Showed high
sequence homology with PrMADS1 (Yao et al. 1999)
MdMADS8 More strongly expressed in fruit than flower buds. Highest expression in young fruit. Most
strongly expressed in core and cortex; weak expression in skin. Showed high sequence
homology with AGL2, AGL4, MdMADS1 and MdMADS9 (Yao et al. 1999)
MdMADS9 More strongly expressed in fruit than flower buds. Highest expression in younger fruit. Most
strongly expressed in core and cortex; weak expression in skin. Showed high sequence
homology with Arabidopsis AGL2 and AGL4 (Yao et al. 1999)
MdMADS10 More strongly expressed in fruit than flower buds. Highest expression in young fruit. Only
expressed in core. Showed high sequence homology with Arabidopsis AGL11 (Yao et al.
1999)
MdMADS11 High expression in both fruit and flower buds. Preferentially expressed in fruit after
pollination. Highest expression in young fruit. Evenly expressed in all three fruit tissues.
Showed high sequence homology with Arabidopsis AGL6 (Yao et al. 1999)
MdMADS12 Isolated from leaf tissue but has significant homology with the Arabidopsis floral identity
gene AP1. Expressed at similar levels in leaves, vegetative shoots and floral tissues. May play
a role in the transition from juvenile to adult stage (van der Linden et al. 2002)
MdMADS13 Isolated from leaf tissue but has significant homology with the Arabidopsis floral identity
gene AP3. Mainly expressed in petals and stamens (van der Linden et al. 2002, Kitahara et al.
2004)
MdMADS14 Isolated from leaf tissue but has significant homology with the Arabidopsis floral identity
gene AGAMOUS. Preferentially expressed in carpels. Possible orthologue of
SHATTERPROOF (van der Linden et al. 2002)
MdMADS15 Isolated from leaf tissue but has significant homology with the Arabidopsis floral identity
gene AGAMOUS. Highly expressed in stamens and carpels (van der Linden et al. 2002)
MdPI Shows high amino acid sequence identity with Arabidopsis PI. A retrotransposon insertion
was identified that abolished expression of the gene and probably led to parthenogenic fruit
development (Yao et al. 2001)
 13

Several cDNA libraries have been developed to identify genes associated with pollination
and apple fruit development (Dong et al. 1997, Dong et al. 1998b, Sung et al. 1998). Mdh3
encoding a Phalaenopsis O39-like homeodomain protein was found to be expressed in apple
ovules and may initiate the program of ovule development (Dong et al. 1999). A homologue
of mammal DAD1 (defender against cell death 1) was cloned that is expressed after flower
pollination and during senescence of leaves, petals and fruit (Dong et al. 1998c). A gene
encoding polygalacturonase-inhibiting protein (PGIP) was isolated that has two peaks of
expression during apple maturity and is activated by wounding and fungal infection (Yao et
al. 1999).

A number of MADS-box genes have been cloned and characterized from apple (Table 1.4).
These genes produce transcription factors which play an important regulatory role in the
development of floral meristems in all plants. To date, one of the most interesting MADS-
box genes that has been cloned is a mutation of MdP1 caused by a retrotransposon insertion,
which abolishes gene expression and leads to parthenocarpic fruit development (Yao et al.
2001).

Homeobox genes have also been identified in apple that encode homeodomain proteins
which are transcription factors that regulate a number of developmental processes. Watillon
et al. (1997) identified three KNOTTED1 (kn1)-like homeobox genes, KNAP1 – 3.
Transcripts from KNAP3 accumulated in a wide rang of vegetative and reproductive organs,
while mRNAs from KNAP1 and KNAP2 were present primarily in elongated parts of stems.
Sakamoto et al. (1998) isolated two additional (kn1) -like homeobox genes, APHB1 and
APHB2. APHB1 are expressed in shoot apical tissues, stems and flowers but not mature
leaves and fruit. APHB2 is expressed in all organs involving mature leaves and developing
fruit. Another homeobox gene, MDH1, was isolated from developing fruit, flowers and
leaves of apple that has a homeodomain similar to BEL1 which is involved in ovule
development in Arabidopsis (Dong et al. 2000).

1.5.3 Fruit quality

Many of the traits associated with fruit quality are quantitatively inherited including flavor,
shape, size and texture (Table 1.3). Skin color is also quantitatively inherited, but the number
of major genes regulating it may be limited. Anthocyanin stripes are regulated by a single
dominant gene in 'Cox’s Orange Pippin' (Klein 1958). It has been proposed that three,
dominant major genes regulate color (A, B and C) (Lespinasse et al. 1988). Yellow is
produced by one dominant allele, red if more than two dominant alleles are present. The
yellow cream flesh color of 'Cox’s Orange Pippin' is dominant (Alston 1981). Russett is
regulated by a single dominant gene, with numerous interacting polygenes (Alston and
Watkins 1973), yet some russetted clones must only have the mutation in the LI as they do
not transmit this trait to their offspring. A RAPD marker has been identified for fruit skin
color (Cheng et al. 1996). Recently the myB transcription factor has been suggested to
regulate apple red fruit color (Espley et al. 2007, Takos et al. 2006).

A number of genes have been identified and cloned that influence fruit quality. An allele of
the 1-methycyclopropene softening slower gene (Md-ACS1) was found that was significantly
associated with softening in a screen of 35 cultivars (Oraguzie et al. 2004).

Genes associated with anthocyanin biosynthesis were cloned from apple fruit skin, and
 14

cDNAs were identified that encode flavanone 3-hydroxylase (F3H), dihydroflavonol

reductase (DFR), anthocyanidin synthase (ANS), and UDP-glucose:flavonoid 3-O-
glucosyltransferase (UFGT). Each gene was found to be a member of a multigene family.
The mRNAs of these genes were detected preferentially in the skin tissue and were light
induced. The transcripts were more abundant in the skins of cultivars with red skin than non-
red, indicating that these genes have major roles in determination of apple skin color.

The expression of six genes (PAL, CHS, CHI, F3H, DFR and ANS) involved in anthocyanin
production was also studied during flower development (Dong et al. 1998a). Maximum
accumulation of all 6 RNAs was highest during early flower development and dropped
drastically after petal expansion. Blocking of UV or natural light greatly reduced expression
of these six genes and inhibited anthocyanin production, and after re-exposure to light, white
flowers were not able to resynthesise anthocyanins.

In a study of the genetics of commonly found storage disorders, Volz et al. (2001) found high
heritability for soft scald and superficial scald, moderate heritability for water core, and low
heritability for external pit, internal pit, brown heart, breakdown and chilling injury. Two of
the genes that are likely associated with superficial scald have been cloned from apple, hmg1
and hmg2 (Pechous and Whitaker 2002, Rupasinghe et al. 2001). These genes encode 3-
hydoxy-3-methylglutaryl coenzyme A reductase (HMGR) which catalyses the synthesis of
mevalonate from HMG-CoA. Superficial scab is thought to be caused by the oxidation of α-
farnesene in apple skin and α-Farnesene is produced in the mevlonate pathway. A gene
encoding (E,E)- α-farnesene synthase gene (AFS1) has also been cloned that uses farnesyl
diphosphate as a substrate.

Boss et al. (1995) identified the gene for a full length polyphenol oxidase (pAPO5) from a
'Granny Smith' fruit peel cDNA library whose expression was induced by wounding and was
elevated in peel with superficial scald. Polyphenoloxidase (PPO) is thought to play an
important role in browning after the wounding of apples. Kim et al. (2001) cloned pPPO5
from 'Fuji' and identified another full length PPO gene, pMD-PPO2, which shared about 55%
identity. MD-PPO2 was expressed in all stages of flower development, while the APO5
transcript was detectable only in late anthesis. Both genes were expressed during early fruit
ripening; however, only APO5 was significantly induced by wounding.

Overall, flavor is a quantitative trait, but some of its individual components have been found
to be regulated by single genes. The distinct aroma of 'Cox’s Orange Pippin' is the result of a
single dominant allele (Alston and Watkins 1973). A dominant allele also determines
moderate to high acidity, with the specific levels being inherited quantitatively (Nybom 1959,
Brown and Harvey 1971). Resistance to bitter pit was reported to be regulated by two
dominant alleles (Korban and Swiader 1984).

A number of recent studies have shown that the antioxidant capacity of apples can have wide
ranging health effects including the inhibition of colon- and liver- cancer cells (Eberhardt et
al. 2000, Schirrmacher and Schempp 2003). Schmitz-Eiberger et al. (2003) found high
variability among cultivars in ascorbic acid and phenolic compounds. 'Topaz', 'Berlepsch',
'AW 93', 'Golden Delicious', 'Rubinette', 'Braeburn' and 'Honeycrisp' had among the highest
levels of ascorbic acid, while the highest levels of phenolics were found in 'Scesterimuher',
'Bortlinger', 'Bohnapfel' and 'Dulmener Rosenapfel'. Lee et al. (2003) found 'Rhode Island
Greening' to have unusually high levels of antioxidants, while Lata (2007) documented the
 15

effect of cultivar and seasonal variation. Davey et al. (2006) identified QTL affecting vitamin
C in apple using the mapping population of 'Braeburn' x 'Telamon'.

Two types of genes associated in hormone biosynthesis have been cloned and isolated.
Kusaba et al. (2001) isolated a cDNA encoding gibberellin (GA) 20-oxidase that was mainly
expressed in immature seeds. Wegrzyn et al. (2000) cloned an α-amylase gene from apple
fruit that was transiently upregulated during low temperature exposure. Stanley et al. (2002)
also isolated several α-amylase genes from apple and Arabidopsis that they suggested might
be targeted to different compartments within the cell (cytosol, secretory pathway and plastid).

Genes for ACC-synthase, ACC-oxidase and polygalacturonadase (PG) have been cloned and
characterized from apple (Dong et al. 1991, Castiglione et al. 1999) and the promoter
sequences of the genes for ACC-oxidase and PG have been characterized (Atkinson et al.
2005). Castiglione et al. (1999) examined restriction products across 12 Malus species and
found two allelic forms of a gene for ACC-oxidase but very little variability in a gene for
ACC-synthase. They suggested that the two allelic forms of ACC-oxidase might control the
rate of ethylene synthesis and could be used in marker assisted selection. Harada et al. (2000)
found a specific allele of ACC-synthase (Md-ACS1-2) that was associated with low levels of
ethylene production in a screen of 35 cultivars. Atkinson et al. (1998) examined the
expression of PG and ACC-oxidase mRNAs and detected them earlier in 'Royal Gala' apples
relative to internal ethylene concentration than 'Braeburn' and 'Granny Smith'.

Tao et al. (1995) identified a cDNA encoding NADP-dependent sorbitol-6-phosphate

dehydrogenase (S6PDH) from apple and found that protein levels were highest in mature
fruit. Sorbitol is the end product of photosynthesis in apple. Tao et al. (1995) incorporated the
gene into tobacco and found that sorbitol levels were positively correlated with S6PDH
activity levels. Yamada et al. (1998) identified a full-length cDNA for NAD-dependent
sorbitol dehydrogenase (NAD-SDH) and showed that the mRNA is expressed in mature
apple fruit.

A number of plant-derived allergens have been identified and placed into specific groups,
including pathogenesis-related proteins (PR), seed storage proteins and structural proteins
(Hoffmann-Sommergruber 2002). Representatives of three families of PR genes have been
cloned and characterized in apples including: 1) Mal d 2, producing a thaumatin-like protein
(Krebitz et al. 2003), 2) Ypr10, producing a intercellular protein with unknown enzymatic
action (Puhringer et al. 2000), and 3) Mal d 3, producing a lipid transfer protein (LTP) (Diaz-
Perales et al. 2002). The promoter of Ypr10 is both stress- and pathogen-inducible, and the
product of Mal d 2 has anti-fungal properties. Gao et al. (2005a and b) cloned and mapped
Mal d 1 and also mapped Mal d 2 and 4. The location of allergens was studied by Marzban et
al. (2005). Mal d 1 and 2 were distributed in peel and flesh, while Mal d 3 is restricted to the
peel.

Breeding programs are assessing low allergenicity as a breeding objective. Gao et al. (2005a)
cloned and mapped the major apple allergen Mal d 1, and then studied Mal d 3 (2005b).
Carnes et al. (2006) demonstrated differences in antigenic and allergenic profiles for 10
different apple varieties and found significant variation in content of Mal d 3.
 16

1.6 Crossing and evaluation techniques

1.6.1 Breeding systems

Most apples require cross-pollination; in the orchard, pollination is carried out primarily by
bees. Self-fertility is limited in apple by gametophytic self-incompatibility, where the growth
of self pollen tubes is prevented by cytotoxic proteins that are produced in the stigmatic
tissue. Attack is avoided if specific inhibitors of these proteins are expressed. The style-
encoded toxic proteins are RNases which are produced by the S-gene. The pollen-expressed
inhibitors have not been identified. Allele-specific PCR primers have been developed to
selectively amplify and identify individual S-alleles, and 28 S-alleles have been cloned and
identified in 150 diploid and triploid European, American and Japanese cultivars (Broothaerts
et al. 2004a). Three S-alleles (S2, S3 and S9) are very common and seven are very rare (S4, S6,
S8, S16, S22, S23 and S26). The allelic composition of the most widely grown cultivars are:
Delicious (S9S28), Golden Delicious (S2S3), Granny Smith (S3S23), Fuji (S1S9) and Gala (S2S5).
S-RNase analysis has been used to identify the parents of Japanese cultivars (Kitahara et al.
2005, Matsumoto et al. 2006). Broothaerts (2003) suggests that some S-alleles be renumbered
based on new research.

1.6.2 Pollination and seedling culture

The blossom is typically composed of five petals, five sepals, about 20 stamens and a pistil
with five styles. The flowers are borne in cymose clusters on short pedicels. Each ovary
generally has five carpels with two ovules each, resulting in a total of 10 seeds (although
some varieties can have up to 18 seeds).

For pollen collection, flowers are gathered at the balloon stage before petal expansion.
Blossoms can be collected from rooted plants in the field or greenhouse, or flowers can be
forced in the greenhouse by cutting flowering shoots and holding them in water. Anthers are
first removed from flowers by passing them over a screen and then allowed to dehisce
overnight in containers such as Petri plates or “paper boats”. The dry pollen is ready to be
used directly in crosses and remains viable for several days at room temperature. It can
remain viable for several weeks if refrigerated under low relative humidity. For long term
storage, pollen can be held for at least a year at -15 ºC in loosely stoppered vials in a
desiccators with calcium chloride.

To emasculate flowers, fingernails or scissors are generally used to remove sepals, petals and
stamens at the balloon stage. Pollinations are generally made soon after emasculation,
although flowers are sometimes re-pollinated one day later if conditions are thought to be too
cool for normal pollen germination and tube growth. Flowers can be successfully fertilized
over a period of several days. Generally, two flowers per cluster are emasculated and the rest
are removed. There is no need to cover the emasculated flowers after pollination, as insects
do not visit flowers without stamens and petals (Visser 1951). Some breeders do not
emasculate at all and rather rely on the self incompatibility system to prevent self
fertilization. In this case, flowers are bagged to prevent contamination. Keulemans et al.
(1994) discussed the effect of number of flowers pollinated on fruit set in crosses.

Pollen is often placed on the stigmas after dipping a small brush into vials or Petri plates of
 17

pollen. Pencil erasers or fingertips are also sometimes used to transfer pollen. After each
cross, the pollination vehicle is washed or dipped into 95 % alcohol and allowed to dry to
prevent cross contamination.

Several other techniques are sometimes employed to eliminate the emasculation step. Small
trees can be enclosed in a bee-proof structure with a bouquet of open flowers of the desired
parent and honeybees. Bouquets of potential parents with dominant marker genes can also be
placed adjacent to several recipients in a field, and the desired hybrids can be identified in the
seedling blocks. Seed can also be collected from orchards that contain two cultivars of
interest.

Most breeders attempt to get at least 200 to 300 seeds per cross, although thousands of seeds
are sometimes generated of crosses that are thought to have great commercial potential or
when seedling screening is planned using inoculation or markers. About 50 to 100
pollinations are typically required to generate a few hundred seeds, but if flowers are
emasculated, the general rule is that on average one flower produces one seed due to damage
from emasculation. The number of crosses made varies greatly depending on program
objectives, available resources and philosophy of the breeder, but can range from 5 to 50. If
both parents are heterozygous for pale green lethal (Way et al. 1976), dwarfing genes (Alston
1976) or sub-lethals linked to the Vf gene (Gao and van de Weg 2006) then greater numbers
are needed, due to the expected 25% loss due to lethals and dwarfs.

Apple seeds must be stratified in the cold for successful germination. Seeds can be left in the
fruit at slightly above freezing temperatures to naturally after-ripen, but when this is done,
molds often become a problem. More commonly, seeds are harvested just before the fruit
reach maturity, but late enough for the seed coats to have become dark-brown. The seeds are
then held in the cold at 3 – 5° C for 60 to 80 days in plastic bags containing moist filter paper
or peat moss. Length of stratification may vary depending on the genetic background.
Thomsen and Eriksen (2006) found that two Malus species (Malus sargentii and M. sieboldii)
differed in their response to pretreatments and stratification temperatures. When their radicals
begin to emerge, the seeds are transplanted into pots or trays at 1-2 cm depth. They are
maintained under greenhouse conditions for about 60 days until they become 30 – 45 cm tall,
and then they are planted in a nursery or moved outside in larger pots. Plants are also
sometimes pre-selected before field planting for plant vigor, large mature-phase leaf type and
growth habit/architecture. Often seedlings are inoculated with scab in the greenhouse,
particularly if at least one parent is known to be resistant; this technique can reduce the
progeny population by 50 - 80% (Janick et al. 1996).

1.6.3 Evaluation techniques

The juvenile period without fruit varies from 3 to 10 years, depending on genotype and
growth environment. A number of techniques have been used to shorten the duration of the
juvenile period, including shoot pruning, root pruning and bark ringing (Janick et al. 1996);
however, most of these are tedious and difficult to utilize with large populations. Probably
the most helpful approach is to maintain active growth in the greenhouse before planting and
throughout the entire evaluation period. Many programs will graft seedlings onto dwarfing
rootstocks (M9 most common, also Bud 9 and EMLA 27) either in the first or second year.
Dwarfing rootstock promotes more precocious flowering and saves space, but the use of
rootstocks adds to the cost of the program. The rootstocks must be from virus-indexed stock
 18

to avoid infecting scions.

Field selection is generally performed in three stages: Phase 1 – Genotypes are replicated
only once whether grafted or not. If they are on their on own roots, spacing is usually 1.5 to
2.0 m in row and 5.0 to 7.0 m between rows. If they are on dwarfing rootstocks, they are
usually spaced 0.6 to 1.0 m in the row and 4.0 to 5.0 m between rows. Phase 2 - the most
promising seedlings are cloned by grafting on dwarfing rootstock (M9 most common);
usually 4 to 6 trees are produced and are planted as a single unit or split into two replications.
The replications are usually evaluated at only one location but sometimes they are planted at
two sites. Phase 3 – Pre-commercial testing is conducted with multiple trees (10-50 is
common) placed at many locations across the world (10 to 20 would not be uncommon).
Virus indexing (and thermotherapy if needed) is usually performed at this stage on the most
promising selections.

More breeding programs are starting to evaluate what quality traits are most important to its
consumers. Fruit quality, aroma, consistent and high soluble solids, a range of acidity, high
juiciness, crispness and non-browning flesh are desirable as are methods to quantify these
traits. Sensory testing is becoming a part of many programs, as are studies of quality
components (Harker et al. 2006).

1.7 Biotechnological approaches to genetic improvement

1.7.1 Genetic mapping and QTL analysis

Molecular markers have been linked to a number of monogenic traits in apple (Tartarini and
Sansavini 2003). The most work has been done on the Vf gene for scab resistance, where over
40 markers have been identified. Markers for the other scab resistance genes have also been
developed by many groups and include Vh from Russian seedling R12740-7A of M. sieversii
(Hemmat et al. 2002, Bus et al. 2005a and b, Boudichevskaia et al. 2006), Vm (Cheng et al.
1998, Patocchi et al. 2005), Va and Vb (Hemmat et al. 2003, Erdin et al. 2006), Vd (Tartarini
et al. 2004), Vbj (Gygax et al. 2004) and Vg (Durel et al. 1999, Calenge et al. 2004). Gessler
et al. (2006) reviewed the literature in this area from type of resistance through gene
pyramiding.

Markers have also been linked to the pest resistance genes Sd1 for Dysaphis devecta, and Er1
and Er2 for Eriosoma lanigerum (Table 1.2). A few markers have also been linked to genes
regulating morphological traits including the columnar habit (Co), fruit color (Rf) and fruit
acidity (Ma) (Table 1.3). Recently a cDNA/AFLP approach was used to identify a gene that
contributes to lowering of fruit acidity (Yao et al. 2007).

A number of apple linkage maps have been published using several different sets of parents:
'White Angel' x 'Rome Beauty' (Hemmat et al. 1994), 'Wijcik McIntosh' x NY 75441-58
(Conner et al. 1997), 'Prima' x 'Fiesta' (Maliepaard et al. 1998), 'Iduna' x A679-2
(Gianfranceschi et al. 1998), 'Fiesta' x 'Discovery' (Liebhard et al. 2002) and 'Telamon' (a
columnar genotype) x 'Braeburn' (Kenis and Keulemans 2005). The one with the greatest
genome coverage and marker density is that of Liebhard et al. (2002), with 475 AFLPs, 235
RAPDs, 129 SSRs and 1 SCAR marker. Two parental maps were constructed that spanned
1,140 and 1,450 cM, respectively. While their map was composed primarily with normally
 19

segregating markers, several linkage groups were found to carry groups of markers with the
same distorted ratios. The highly transferable SSR frame of this map will make it a useful
starting point for future Malus mapping projects.

In the first QTL study in apple, randomly amplified polymorphic DNAs (RAPDs) were used
to locate genes associated with juvenile tree growth and development in the cross between
the columnar mutant 'Wijcik McIntosh' and a standard form, disease resistant selection NY
75441-58 (Conner et al. 1998). One to eight QTL were identified for a number of traits
including height increment, internode number, internode length, base diameter, branch
number and leaf break. The amount of variation explained by regression on individual loci
ranged from 3.9 to 24.3, with an average of 7%. Most QTL were significantly associated with
a trait in only in one or two years.

Several groups in Europe have been especially busy mapping the QTL associated with
resistance to apple scab in various linkage groups (LGs). The cross of 'Prima' x 'Fiesta' and
other related F1 progenies have been used to identify major genes associated with resistance
in the D.A.R.E. project (Durable Apple Resistance in Europe) (Durel et al. 2002 and 2003).
The major genes for scab resistance Vg were found on LG 12. Several different NBS-type
resistance gene analogues were clustered at bottom of LG 5 and at the top of LG 10.
Numerous QTL for partial scab resistance were identified that mapped to four genomic
regions. Most of these QTL were race specific with a few exceptions that included a QTL on
LG 2 for resistance to races 6 and 7, and a QTL on LG 17 for resistance to races 1 and 6. A
major non-race-specific QTL was identified near an NBS-analog cluster on linkage group LG
10. Three major genes for powdery-mildew resistance were also identified by bulked
segregant approaches, and one of them on LG 2 was located in the same region as scab
resistance.

Vinatzer et al. (2004) used the inverse polymerase chain reaction and simple sequence
repeats to identify BAC clones containing the apple scab resistance gene Vf and found the
gene in scab-resistant accessions of Malus micromalus and 'Golden Gem' of M. prunifolia.,
which were previously not known to carry this gene. They also found a mistake in the
published pedigree of the Vf cultivar 'Florina' by comparing SSR patterns of its presumed
progenitors to characterized ones.

Five apple progenies were used in the D.A.R.E. project to identify QTL with broad spectrum
of resistance towards a wide range of strains of the fungus (Durel et al. 2004). It was verified
that four major genomic regions exist that carry resistance to multiple strains of the fungus,
with a QTL region on LG 17 carrying the widest spectrum of resistance. Several other
linkage groups carry QTL or major resistance genes to specific isolates.

Resistance to apple scab was also mapped in the cross 'Fiesta' x 'Discovery' (Liebhard et al.
2003a). Eight genomic regions were identified in this study, with six conferring resistance to
leaf scab and two to fruit scab. The amount of variation attributed to the various genes ranged
from 4 – 23%, with all but one of the QTL being present across multiple years and locations.
Two of the scab resistance QTL reported by Durel et al. (2002) were located on the same
linkage groups (LG 10 and 17) and two were not. While 'Discovery' showed more resistance
to scab in the field, the most QTL were identified in the more susceptible parent 'Fiesta'. This
may indicate that the resistance genes in 'Discovery' are largely homozygous and can not be
detected because they do not segregate.
 20

Resistance gene homologues have been mapped in two segregating populations, 'Fiesta' x
'Discovery' (Baldi et al. 2004) and 'Discovery' x TN10-8 (Calenge et al. 2005). The gene
homologues are widely distributed across the genome, but often reside in clusters. A high
number of the markers mapped close to major genes or QTL for resistance to scab and
mildew. Research on nucleotide binding site (NBS)-encoding resistance gene homologs
(RGHs) among the Rosaceae revealed synteny of a geneomic region that encompasses
powdery mildew resistance locus among Malus, Prunus and Rosa (Xu et al. 2007).

Progeny from the cross of 'Prima' x 'Fiesta' were used to detect QTL associated with physical
and sensory descriptors related to fruit flesh firmness (King et al. 2000 and 2001). Significant
QTL were identified on nine linkage groups that were associated with firmness, stiffness,
slow breakdown, crispness, granularity, hardness, juiciness, sponginess and overall liking.
Considerable variability was noted across years and sites for penetrometer and acoustic
resonance readings, and the presence of the QTLs associated with these traits was also highly
variable. A highly significant QTL was detected on LG 16 for firmness, crispness, juiciness,
sponginess and overall liking. QTL for penetrometer or acoustic resonance measures were
not detected in this region, although it did map with Ma, the malic acid gene (Maliepaard et
al. 1998). Several significant QTL associated with firmness and juiciness on linkage group
LG 1 are found in proximity to the locus Vf, originating from the scab resistant crab apple
Malus floribunda.

1.7.2 Regeneration and transformation

The first apple transformation was done by James et al. (1989) using Agrobacterium
tumefaciens. Since this seminal study, much work has been conducted to improve the
efficiency of gene transfer and regeneration and a wide array of cultivars have been
transformed and regenerated (Hammerschlag 2000, Brown and Maloney 2003). Particle
bombardment of apple leaf explants has received much less emphasis, although Gercheva et
al. (1994) developed protocols for 'Royal Gala'.

Several genes have been inserted into apple to provide resistance to fungal diseases, although
their efficacy in generating pathogen resistance has not yet been published. The stilbene
synthase gene from grapes and polygalacturonase-inhibiting protein from kiwi were
transferred to 'Holsteiner Cox' and 'Elstar' (Szankowski et al. 2003). The antimicrobial
peptide gene A1-AMP was incorporated into 'Jonagold' (Broothaerts et al. 2000a). Transgenic
lines of 'Orin' and 'JM 7' have been selected with genes encoding chitinase, glucanase and
sarcotoxin (Soejima et al. 2000).

Engineering resistance to apple scab has been the focus of several laboratories. Belfanti et al.
(2004) isolated the HcrVf2 gene for apple scab resistance from wild Malus floribunda and
found it confers resistance in transgenic 'Gala'. Bolar et al. (2001) found that expression of
endochitinase from the biocontrol fungus Trichoderma atroviride increased resistance to
apple scab in transgenic 'Marshall McIntosh'. In other work, this group inserted genes for
both endochitinase and exochitinase from T. atroviride, and found they had a synergistic
activity against the pathogenic fungi Venturia inaequalis (Bolar et al. 2000). Chevreau et al.
(2001) inserted the gene for puroindoline-b from wheat in both susceptible and resistant
apple cultivars, but did not report on whether resistance was achieved.
 21

A considerable amount of work has been undertaken to develop fire blight resistant apples
through genetic engineering (Aldwinckle et al. 2003, Norelli et al. 2003). Initial efforts
focused around transferring genes for anti-microbial proteins to apple, including attacin E,
avian lysozyme and the cecropin analogs, SB-37 and Shiva-1. The highest levels of
resistance were found in the attacin-transgenics, but the cecropin- and avian lysozyme
analogues were also effective. In long term field trials, fruit from transgenic lines of 'Royal
Gala' and 'Galaxy' containing the anti-microbial genes was indistinguishable from the fruit of
non-transformed trees.

In the most recent attempts to engineer resistance to fire blight, antimicrobials proteins have
been introduced into apple that act directly against the pathogen, Erwinia amylovora (Norelli
et al. 2003). Two genes are being investigated: 1) the harpin gene (hrpN) from E. amylovora,
which produces an effector molecule that induces resistance when applied to apple flowers,
and 2) genes for DspE-interacting kinases, which interfere with the DspE pathogenesis factor
from E. amylovora. The gene for NPR1 protein (MpNPR1) has also been studied, whose
homologue in Arabidopsis is thought to be a key regulator in the induction of disease
resistance.

In other work on pest resistance, Viss et al. (2003) developed transgenic 'Jonagold' that was
resistant to crown gall disease, by inserting genes designed to express double-stranded RNA
from the iaaM and ipt sequences of the ocogenes of Agrobacterium tumefaciens. These genes
are responsible for the excessive hormone production that leads to gall formation. Markwick
et al. (2003) found that apple plants of 'Royal Gala' expressing biotin-binding proteins were
resistant to the lightbrown apple moth. Yao et al. (1995) produced 'Royal Gala' plants
resistant to the herbicide GleanTM by transforming them with pKILI110, a mutant of the
Arabidopsis acetolactate synthase gene.

Several transgenes have been shown to have significant effects on apple growth and
development. Early flowering was induced in transgenic apple when BpMADS4 from silver
birch (Betula pendula Roth.) was overexpressed (Flachowsky et al. 2007). Holefors et al.
(2000) found the Arabidopsis phytochrome B gene to reduce shoot, root and plant dry
weights in transformed M26 rootstock. Bulley et al. (2005) isolated an apple GA 20-oxidase
gene and inserted it into 'Greensleeves' in the sense and antisense orientations and produced
dwarf lines with both constructs. Application of GA3 restored the internode length and
number of these transgenic lines, and the scion remained dwarfed after grafting to normal
rootstocks. Atkinson et al. (2002) found overexpression of polygalaturonase in transgenic
'Royal Gala' led to a range of novel phenotypes including silvery colored leaves and
premature leaf shedding. Mature leaves also had malformed stomata that effected water
relations and lead to brittle leaves.

The rol A, B, C genes from Agrobacterium rhizogenes are known to influence hormone
metabolism and root development during infection, and as such have been tested as a means
of influencing apple growth and development. The integration of the rolA gene into the
genome of apple rootstock A2 reduced plant height and shortened internodes (Zhu et al.
2001a). The transformation of apple rootstock M.9/29 with the rolB gene reduced node
number and stem length, but not relative growth rate (Zhu et al. 2001b). Root percentage and
root number was increased in shoots of Jork 9 rootstock and the apple scion 'Florina' through
the insertion of rolB (Radchuk and Korkhovoy 2005, Sedira et al. 2001). Introduction of rolC
into the apple rootstock 'Marubakaidou' produced four different phenotypes in transformants:
 22

a group with reduced height and shortened intervals, a group with reduced height but normal
internode lengths, a group with normal height with shortened intervals and a group that was
phenotypically similar to control plants (Igarashi et al. 2002).

To reduce browning, Murata et al. (2000) produced transgenic 'Orin' apples carrying the
antisense of polyphenol oxidase (PPO). The approach worked, as some of the transgenics had
significantly lower levels of PPO than non-transgenic shoots and less browning. Broothaerts
et al. (2000b) developed a spectrophotometric assay to rapidly screen PPO activity in apple.

Transgenic apple trees have been produced that possess extra copies of the endogenous S-
gene controlling self-incompatibility in apple to induce self-fertility (Broothaerts et al. 2004a
and b). In controlled self and outcrosses over a 3-year-period, the transgenic lines had normal
levels of fruit and seeds after selfing, while the control plants had significant reductions. The
self-fertile transgenic type was associated with an absence of pistil S-RNase proteins.

Gilissen et al. (2005) silenced the major allergen Mal d 1 using the RNA interference
approach. Allergen levels were reduced but not eliminated.

Transgenic approaches are adding to our knowledge of flavor and ethylene responses.
Dandekar et al. (2004) examined the effect of down-regulation of ethylene biosynthesis on
fruit flavor complex in apple fruit. In related studies, the relationship of ethylene biosynthesis
to volatile production, related enzymes, and precursor availability in apple peel and flesh
tissues was studied by Defilippi (2005a and b) who found that alcohol acyltransferase, a rate
limiting step for ester biosynthesis important in aroma, is regulated by ethylene.

James et al. (2001) and his group (Gittins et al. 2000, 2001 and 2003) have studied the ability
of a number of heterologous and homologous promoters to drive expression of β-
glucuronidase in tissues of apple. They found the ribulose-1,5-bisphosphate
carboxylase/oxygenease small-subunit promoter (RBCS3C) from tomato and SRS1P from
soybean to primarily drive activity in vegetative tissues of apple that had chloroplasts. The
SRS1P promoter was regulated by light, while RBCS3C was not. They also found the extA
promoter from rape to be very active in all apple tissues, even though its activity is root-
specific in its own species. The vascular tissue promoters, rolC from Arabidopsis rhizogenes
and COYMV from the Commelina yellow mottle virus were found to have localized
expression in structural tissues. The group has also been active in identifying ethylene
inducible promoters from apple.

Cisgenic approaches, using genes from apple in transformation, is discussed by Jacobsen and
Schouten (2007).

1.7.3 Genomic resources

Mining of existing apple EST information, such as the studies of Newcomb et al. (2006) and
Park et al. (2006), and the use of microarrays (Lee et al. 2007, Pichler et al. 2007) promises
to expand our knowledge of many genes important in the genetic improvement of apple. The
development of public databases such as the GDR (Genome Database for the Rosaceea; Jung
et al. 2004) and the European HIDRAS AppleBreed (Antofie et al. 2007) also offer excellent
prospects for enhanced collaboration amongst breeders, bioinformatics researchers and those
 23

involved in molecular biology. In the GDR database alone, over 50,000 ESTs are available
from several species, tissues and developmental stages.

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