1. Breeding Potential in Danish Apple Cultivars:
Genetic Diversity and Genome-Wide Association Mapping
of Fruit Quality Traits
PhD Thesis
Bjarne Larsen
Genetic Diversity and Genome-Wide Association
Mapping of Fruit Quality Traits
DEPAR TM ENT OF PLAN T & E N V IR O N ME N TAL SCIENCES
FAC ULT Y OF SC IEN CE · U N IV E R S IT Y O F CO PENHAGEN
PHD T H E SI S 2017 · ISB N 978-87-93476-72-1
BJARNE L ARSEN
Breeding Potential in Danish Apple Cultivars:
Genetic Diversity and Genome-Wide Association Mapping of Fruit Quality Traits
Breeding Potential in
Danish AppleCultivars:
PHD THESIS 2017 | BJARNE LARSEN
BJARNELARSENBreedingPotentialinDanishAppleCultivars
university of copenhagen
faculty of science
2.
3. U N I V E R S I T Y O F C O P E N H A G E N
F A C U L T Y O F S C I E N C E
Breeding Potential in Danish Apple Cultivars:
Genetic Diversity and Genome-Wide Association Mapping
of Fruit Quality Traits
PhD Thesis
Bjarne Larsen
5. 2
Vel! Ræk mig da, o Efteraar,
en Gravensten som smager
af Bækken ved min Faders Gaard
og Mulden i hans Ager
Ludvig Holstein, 1915
6.
7. 3
Preface
This PhD has been carried out at the Department of Plant and Environmental Sciences,
University of Copenhagen (UCPH). Here, the research has been carried out as a collaborative
work between the three sections, Organismal Biology, Plant and Soil Sciences and Crop
Sciences under the supervision of Marian Ørgaard, Carsten Pedersen and Torben Bo Toldam-
Andersen. The thesis has been submitted to the PhD School of The Faculty of Science,
University of Copenhagen.
The thesis includes a short introduction followed by two introductory chapters, a discussion
and a conclusion. They provide a short overview of the research project in context of reported
knowledge and results from present study. The thesis furthermore includes four manuscripts,
of which one is published, two are accepted for publication and one is in preparation. An
abstract outlining the project was published in an abstract booklet for The Annual Plant Biotec
Denmark Meeting 2016 together with a poster presentation. Another poster outlining major
research goals and preliminary results was presented at The XIV Eucarpia Symposium on
Fruit Breeding and Genetics 2015. Project goals and primary results have been
communicated to the public during open door days in University greenhouses and at the
Pometum – UCPH.
Most experimental lab work was carried out at Faculty of Science, Frederiksberg Campus,
UCPH. Analysis of fruit flavour volatiles was done by Mikael Agerlin Petersen, Department
of Food Science, UCPH. Analysis of genotyping-by-sequencing data and a genome-wide
association study was performed in collaboration with Sean Myles’ group at Dalhousie
University, Canada during a five weeks research stay in November-December 2015.
8. 4
Acknowledgements
First of all, I am deeply grateful to my supervisors, Marian Ørgaard, Carsten Pedersen and
Torben Toldam-Andersen for their trust, help and warm encouragement.
I am thankful to the Department of Plant and Environmental Sciences for offering me a
PhD scholarship and to Foreningen PlanDanmark for a generous grant.
A high number of colleagues at the Department of Plant and Environmental Sciences have
helped and inspired me. I am thankful to all these people that supported me both during my
PhD-study and the years before.
I am thankful to technicians Vinnie Deichmann, Karen Rysbjerg Munk, Mads Nielsen,
Mette Sylvan and Lisbeth Mikkelsen for support and skilful assistance. I am very grateful
to Niels Jacobsen for comments on the thesis and for continuous inspiration and support. I
am also thankful to fruitful discussions with Jihad Orabi and Maren Korsgaard. The
Pometum staff has provided valuable help. Thanks to Elizabeth Cassidy for comments on
the thesis. I feel delighted for the PhD-students that I got to know during my time at the
Department, especially Gaia, Wibke and Mona.
Great thanks to Sean Myles who warmly welcomed and hosted me during a five weeks
visit at Dalhousie University, Canada. I am thankful to the people that I met and
collaborated with at the research station in Kentville, Nova Scotia, especially Kyle Gardner
and Laura Butler.
I am deeply grateful to Jacob Weiner for fruitful discussions and valuable comments on
manuscripts.
I owe an important debt to Sven Bode Andersen for inspiring and introducing me to the
fascinating world of plant breeding.
Special thanks to the former pometmester Claus Larsen who opened my eyes to the beauty
of Danish apple cultivars.
My deepest gratitude goes to my family. To my mother and father who have been with me
my whole life. They have provided the vital support during my professional career all the
way from the daily work as gardener, during the horticultural studies and PhD work.
Thanks to my sister for always being there for me. My warmest affection goes to Gabriela
who came into my life halfway through the PhD period and to our love, little Ellinor.
9. 5
English abstract
The diversity in plant genetic resources is a prerequisite for genetic improvement of cultivated
crop species. Lack of in-depth characterization and evaluation of gene bank accessions is a
major obstacle for their potential utilization. The Danish apple (Malus domestica L.) gene
bank collection represents an ensemble of cultivars that have never previously been
genetically accessed. The aim of this thesis is to study the genetic structure, affiliation and
overall diversity which should facilitate future conservation management strategies. It may
also contribute with new knowledge for better understanding of the link between phenotypes
and the underlying gene-tic background which is crucial in plant breeding. We found a
considerable genetic diversity in the collection and no genetic structure. We exposed a high
number of accessions in admix and revealed several putative cultivar parentages, never
previously reported. Unique fingerprints were obtained for all accessions except for
distinctive subclonal sports and synonym accessions. The cultivar ensemble was shown to
hold 22% triploid accessions. We developed a new protocol for genotyping S-RNase alleles in
apple and revealed 25 different alleles, including several rare alleles. Using historical gene
bank records, including aroma volatile analysis, sugar and acid data and other fruit- and tree
character records, we established genotype-phenotype relationships, performing a genome-
wide association study. A number of SNP markers are presented that can be used directly for
marker-assisted selection. In addition, we suggest a number of candidate genes involved in
the control of several important fruit quality traits. Future studies and breeding attempts can
therefore benefit from the results, including genetic fingerprints and pedigree reconstruction.
In addition, several of the SNP markers presented can be used directly in selection for specific
traits in breeding lines. However, further characterization and evaluation of additional
important horticultural traits are still needed for upmost utilization of the apple gene bank
collection.
10. 6
Dansk resumé
Genetisk mangfoldighed i planter er en forudsætning for genetisk forbedring og tilpasning af
dyrkede planter. Manglende karakterisering af genbankssamlinger er et stort problem for den
potentielle udnyttelse af genbankernes potentiale. Den danske æblesortssamling på Pometet –
Københavns Universitet er aldrig før beskrevet genetisk. Formålet med afhandlingen er derfor
at beskrive den genetiske struktur og diversitet i samlingen. Formålet er også at undersøge
sorternes afstamning og bidrage med resultater, der kan være med til at forbedre fremtidige
bevaringsstrategier. Resultaterne kan også bidrage med øget forståelse af sammenhængen
mellem bestemte egenskaber og den genetiske baggrund, hvilket er af afgørende betydning i
planteforædling. Vi fandt en stor genetisk variation i de danske æblesorter.
Slægtskabsundersøgelser har påvist forældreskabet for mange æblesorter, der hidtil har været
ukendte. Ved hjælp af genetiske markører har vi lavet unikke DNA-fingeraftryk for sorterne,
der kan hjælpe til identifikation, blandt andet til at identificere genetiske dubletter, der
optræder ”skjult” under andre kultivar navne. Vi fandt at samlingen indeholder 22 %
triploider. Vi udviklede en ny metode til at beskrive S-RNase alleler og fandt 25 forskellige
alleler. Denne variation er afgørende for befrugtning blandt æblesorter. Ved at bruge tidligere
beskrivelser af æblesorternes aroma stoffer, sukker og syre indhold samt forskellige frugt- og
trækarakterer, og kombinere dem med nye DNA data har vi opnået ny forståelse for den
genetiske baggrund for æblers kvalitative egenskaber. Vi præsenterer flere DNA markører,
der er direkte anvendelige til at udvælge nye genotyper i æbleforædling. Vi påpeger
kandidatgener for vigtige egenskaber hos sorterne. Resultaterne bidrager med ny viden, der
kan forbedre og målrette de kommende års æbleforædling og forskning. Fremtidige studier er
dog stadig nødvendige for at kortlægge den genetiske baggrund for mange flere
kvalitetsegenskaber. Vejen er stadig lang, men nødvendig, for at skabe de æblesorter der
imødekommer forbrugernes og avlernes vedholdende krav om forbedrede æblesorter til det
nordiske klima.
11. 7
Manuscripts included
Manuscript I Larsen, B., Toldam-Andersen, T. B., Pedersen, C. & Ørgaard, M.
(2017). Unravelling genetic diversity and cultivar parentage in the
Danish apple gene bank collection
Tree Genetics & Genomes, 13(1): 1-12
Manuscript II Larsen, B., Ørgaard, M., Toldam-Andersen, T. B. & Pedersen, C.
(2016). A high-throughput method for genotyping S-RNase alleles in
apple
Molecular Breeding, 36(3): 1-10
Manuscript III Larsen, B., Pedersen, C., Ørgaard, M. & Toldam-Andersen, T. B.
Danish apple cultivars: Genetic diversity, parentage and breeding
potential
Accepted for publication in Acta Horticulturae
Manuscript IV Larsen, B., Gardner, K., Toldam-Andersen, T.B., Myles, S.,
Migicovsky, S., Ørgaard, M., Petersen, M.A., Pedersen, C. Genome-
wide mapping of fruit quality characters in local Danish apple cultivars
using genotyping-by-sequencing
Manuscript in preparation
8
Manuscripts included
Manuscript I Larsen, B., Toldam-Andersen, T. B., Pedersen, C. & Ørgaard, M.
(2017). Unravelling genetic diversity and cultivar parentage in the
Danish apple gene bank collection
Tree Genetics & Genomes, 13(1): 1-12
Manuscript II Larsen, B., Ørgaard, M., Toldam-Andersen, T. B. & Pedersen, C.
(2016). A high-throughput method for genotyping S-RNase alleles in
apple
Molecular Breeding, 36(3): 1-10
Manuscript III Larsen, B., Pedersen, C., Ørgaard, M. & Toldam-Andersen, T. B.
Danish apple cultivars: Genetic diversity, parentage and breeding
potential
Accepted for publication in Acta Horticulturae
Manuscript IV Larsen, B., Gardner, K., Toldam-Andersen, T.B., Myles, S.,
Migicovsky, S., Ørgaard, M., Petersen, M.A., Pedersen, C. Genome-
wide mapping of fruit quality characters in local Danish apple cultivars
using genotyping-by-sequencing
Manuscript in preparation
12. 8
Table of contents
Introduction ........................................................................................................................................... 9
Project aim and objectives.................................................................................................................. 10
Danish apple germplasm as resource ................................................................................................ 12
Nordic apples .............................................................................................................................................. 12
Gene bank practice ..................................................................................................................................... 13
European apple germplasm ........................................................................................................................ 13
Exploring genetic diversity in the collection .............................................................................................. 14
Tracking down the origin of cultivars ......................................................................................................... 14
Measurement of genetic diversity ............................................................................................................. 15
Apple breeding programmes ...................................................................................................................... 15
Nordic apple breeding ................................................................................................................................ 17
Genome-wide approaches in apple .................................................................................................... 19
Phenome to genome .................................................................................................................................. 19
Genome‐wide association .......................................................................................................................... 19
Genomic selection ...................................................................................................................................... 22
Genomic breeding ....................................................................................................................................... 22
Mapping the Danish collection ................................................................................................................... 22
Biosynthesis of aroma volatiles .................................................................................................................. 23
Main aroma volatiles .................................................................................................................................. 24
Fruit volatile GWAS ..................................................................................................................................... 24
Sugar and acid content ............................................................................................................................... 25
General discussion............................................................................................................................... 26
The Danish apple collection ........................................................................................................................ 26
Selected sports or synonyms? .................................................................................................................... 26
Other Malus accessions .............................................................................................................................. 28
Maintenance of compatibility .................................................................................................................... 28
Danish cultivars in breeding ....................................................................................................................... 29
Breeding for new cultivars .......................................................................................................................... 29
Application of GWAS results ...................................................................................................................... 30
Resolution of the GWAS ............................................................................................................................. 30
Gene banks in a non‐static world ............................................................................................................... 30
Conclusions & perspectives................................................................................................................ 32
References ............................................................................................................................................ 33
13. 9
Introduction
Diversity in germplasm resources is a prerequisite for future crop improvement. Thus,
availability and maintenance of an extensive range of genetic diversity is crucial for present
and future breeding. It is also important for research, teaching and public dissemination.
Proper conservation strategies are needed for continuous availability of diverse germplasm
resources.
A small number of successful cultivars have been used to found major apple breeding
programmes worldwide. Thus, apple breeders work with a rather restricted genepool (Noiton
and Alspach, 1996). The consequences are that present day cultivars are rather vulnerable to
changes in pathogens, pests, climate and customer demands. Broadening of the genetic base
in breeding programmes can be done by including non-utilized genotypes kept in gene banks.
Objective measures of genetic variation are needed to ensure maintenance of a broad diversity
in germplasm collections. For breeders, such data are valuable for choosing genetically
distinct breeding partners. Morphological characters have previously served as markers for
genetic diversity which during later decades has been supplemented or replaced by the use of
molecular markers. Such molecular approaches have been used to describe genetic diversity
in several European apple germplasm collections (Urrestarazu et al., 2016). Methodical
progresses in molecular techniques, however, have led to development of high-density marker
systems for accessing genetic resources. Simultaneously, in the field of QTL mapping, next-
generation high-resolution DNA sequence data have replaced low-density marker systems
(Myles, 2013).
Characterization of gene bank material includes both genotyping and phenotyping. The latter
attempt is crucial in order to identify accessions with traits of interest. However, evaluation of
germplasm accessions based on both genotyping and phenotyping facilitates an increased
awareness and improved usability of conserved resources since it allows pinpointing of
accessions with traits of interest. For conservation management strategies both approaches are
crucial in order to prioritize accessions, define collections and finally describe available
germplasm resources.
10
Introduction
Diversity in germplasm resources is a prerequisite for future crop improvement. Thus,
availability and maintenance of an extensive range of genetic diversity is crucial for present
and future breeding. It is also important for research, teaching and public dissemination.
Proper conservation strategies are needed for continuous availability of diverse germplasm
resources.
A small number of successful cultivars have been used to found major apple breeding
programmes worldwide. Thus, apple breeders work with a rather restricted genepool (Noiton
and Alspach, 1996). The consequences are that present day cultivars are rather vulnerable to
changes in pathogens, pests, climate and customer demands. Broadening of the genetic base
in breeding programmes can be done by including non-utilized genotypes kept in gene banks.
Objective measures of genetic variation are needed to ensure maintenance of a broad diversity
in germplasm collections. For breeders, such data are valuable for choosing genetically
distinct breeding partners. Morphological characters have previously served as markers for
genetic diversity which during later decades has been supplemented or replaced by the use of
molecular markers. Such molecular approaches have been used to describe genetic diversity
in several European apple germplasm collections (Urrestarazu et al., 2016). Methodical
progresses in molecular techniques, however, have led to development of high-density marker
systems for accessing genetic resources. Simultaneously, in the field of QTL mapping, next-
generation high-resolution DNA sequence data have replaced low-density marker systems
(Myles, 2013).
Characterization of gene bank material includes both genotyping and phenotyping. The latter
attempt is crucial in order to identify accessions with traits of interest. However, evaluation of
germplasm accessions based on both genotyping and phenotyping facilitates an increased
awareness and improved usability of conserved resources since it allows pinpointing of
accessions with traits of interest. For conservation management strategies both approaches are
crucial in order to prioritize accessions, define collections and finally describe available
germplasm resources.
14. 10
Project aim and objectives
Overall, the aim of the project is to explore the genetic resources of the Danish apple gene
bank collection kept at the Pometum – UCPH. Characterization of germplasm is done using
DNA-markers. For each accession the goal is to determine the ploidy-level in addition to S-
RNase-alleles that are responsible for incompatibility between cultivars. Genome-wide
association mapping will be used to establish marker-trait associations for aroma volatile
compounds, individual sugars and acids, and various fruit- and tree characters. The findings
should help breeders to pinpoint candidate breeding partners, ensure fertilization
compatibility between genotypes and device QTLs for important fruit quality traits. The
outcome of the project will be a tool for future gene bank management in prioritization of
accessions and will contribute to a better understanding of the breeding history of Danish
apples.
Expected outcome of the thesis:
Reveal the genetic structure and diversity in the Danish apple collection
Expose cultivar parentages, ploidy and synonyms among germplasm accessions
A new protocol for S-RNase allele genotyping and expose S-RNase alleles in the
cultivars
High-resolution mapping and identification of QTL-flanking markers and candidate
genes involved in aroma volatiles formation and other frit quality traits
Previous characterization of the Danish apple collection has demonstrated a large variation
among various fruit- and tree phenotypic characters (Toldam-Andersen et al., 2011), fruit
sugar/acid content and fruit flavour volatile compounds (Varming et al., 2013). However, the
genetic variation in the gene bank collection has not yet been examined. The study includes
the Danish apple collection and a reference set of cultivars mainly from other European
countries. A private tree nursery collection, Assens was also included in order to look for new
unique genotypes. The study will examine the genetic variation to gain new insight in the
genetic structure and diversity of the Danish apple gene bank collection (The results are
described in Manuscript I, III and IV).
Hybridization compatibility between apple genotypes is crucial to ensure fertilization and
fruit set. Compatibility is controlled by an S-locus comprising a number of self-
incompatibility S-RNase alleles (Kobel et al., 1939). Thus, genotypes with different S-RNase
alleles are needed for fertilization. Many commercial cultivars descending from targeted
breeding programmes have low diversity in S-RNase alleles (Broothaerts et al., 2004) which
may cause potential incompatibility problems in future generations. Genotyping S-RNase
alleles have been done in a number of studies using allele specific primers; however, a high
number of PCR reactions are required, and there is a risk that rare alleles are overlooked.
Therefore, we will develop a new protocol for genotyping S-RNase alleles combining
15. 11
general and allele specific primers for PCR. The protocol will be used to genotype S-
RNase alleles in the Danish apple collection (The work is presented in Manuscript II).
Characterization and evaluation of germplasm accessions is crucial for outmost utilization of
gene bank resources. It should finally lead to development of new cultivars with improved
traits. Breeding might benefit from marker-assisted breeding where QTL-flanking markers are
required for selection. Thus, documentation of the QTLs flanking markers is needed which
can be obtained through genome-wide association studies (GWAS). A GWAS was performed
using existing gene bank records for fruit- and tree characters, sugar/acid content and fruit
flavour volatile compounds. The goal was to pinpoint candidate genes for specific fruit
quality traits. Genotyping-by-sequencing will be used to generate genome-wide SNP data
in combination with existing phenotype records for GWAS. Using this approach the aim
is to find QTL-flanking markers for several fruit quality traits and to look for the
underlying candidate genes (Further described in Manuscript IV).
16. 12
Danish apple germplasm as resource
Nordic apples
There is a continuous, ongoing process: new apple cultivars continuously arise simultaneously
with other cultivars that constantly get lost. At present about 300 named apple cultivars with
Danish origin are recognized. The vast majority of these cultivars have emerged as chance
seedlings from open pollinations. Climatic factors like frost resistance have acted as selection
pressure as well as resistance ability to diseases and pests. In addition, a large number of
poeple unfamiliar with genetics have selected for various quality traits. Present day cultivars
have originated during the last three centuries, mostly with unknown parentage. Frequent
reports of the approximate year and locality of origin exist for many cultivars (Bredsted,
1893; Matthiessen, 1913; Pedersen, 1950). The majority of cultivars originate from coastal
regions in the eastern and central part of Denmark, where the climate and soil conditions for
apple cultivation are most beneficial. Cultivars originating from the North and Westernmost
part of Denmark (North and West Jutland) are basically lacking.
Today, cultivars that are released from large-scale international breeding programmes are
adapted to the climate conditions found in the major apple growing regions of the world.
Many of these cultivars are generally not well suited to the maritime climate in coastal
Scandinavia, such as Denmark, where the growing season is relatively short and cool and the
winter temperatures fluctuates around 0 ˚C. Thus, local climatic adaptation is a key breeding
goal for regions where climatic parameters are marginal for apple cultivation (Laurens, 1998).
In Northern Europe, standard apples are di-coloured characterized by juiciness, tartness and
good storability (Sansavini et al., 2004). In Denmark, cultivated apples have traditionally been
dominated by cooking apples. However, new cultivars must meet modern customer
preferences for dessert apples primarily for fresh consumption.
The Danish apple gene bank collection
The largest collection of Danish apple cultivars is kept in the gene bank collection at the
Pometum – UCPH. The Pometum is situated in Taastrup (Zealand, Denmark; 55°40'23.4"N
12°18'29.6"E) where the clonal material is kept as grafted, field cultivated trees. The
collection includes around 800 named apple accessions, of which around 300 are of Danish
origin. For security reasons, a complete copy collection of the Danish cultivars is kept at The
Danish Agricultural Museum, Gl. Estrup. In addition, parts of the collection are kept at The
Open Air Museum, National Museum of Denmark, Lyngby and in private collections at
Blomstergården, Viborg, in Fjordvang Frugtplantage, Otterup and by the society “Rødding -
æblets by i Salling”, Rødding. For further security reasons and to reduce maintenance costs,
cryopreservation for long-term conservation of the germplasm has been trialled (Vogiatzi et
al., 2012), and a minor part of the collection is stored in cryo. The Pometum is a part of the
Nordic Genetic Resource Center, NordGen under the Nordic Council of Ministers that aims to
preserve and utilize locally adapted cultivars and landraces.
17. 13
The Pometum collection was established in 1863 by J.A. Dybdahl. During the first part of the
20th
century a considerable number of cultivars were added to the collection by A. Pedersen
(Pedersen, 1925). He carried out an extensive cultivar sampling and recording throughout
Denmark in order to get an overview of cultivars grown in Denmark. At that time full-grown
trees with a long life-cycle were cultivated solitary or in small orchards at numerous farms,
houses, castles, vicarages and monasteries in Denmark. This practice, which was
characterized by a large variety of cultivars, existed until the Second World War. Hereafter it
was replaced by specialized, high-density orchards with uniform spindle trees including only
a limited number of cultivars. Simultaneously, new cultivars that were adapted to the novel
cultivation practises arose from breeding programmes and gradually replaced traditional
cultivars.
Gene bank practice
Apple germplasm is commonly kept as long-living, grafted trees in orchard collections. The
practice occupies relatively large areas of land over longer periods of time, requires a
relatively large amount of man-hours which makes the maintenance costly. It is therefore
important to make sure that each genotype is represented only once in the collection.
Prioritization ensures that specific alleles are not overrepresented in favour of others. Each
accession should be evaluated on basis of genotype and phenotype in order to justify its
presence in the gene bank. Collection and preservation strategies for endangered genotypes
should continuously be attended (Urrestarazu et al., 2012), however, the genetic value should
be assessed before inclusion in a gene bank collection.
Renewing apple germplasm collections involves a range of procedures such as selection of
scions, grafting, labelling and planting. During these working procedures, there is a risk that
some accessions lose identity and that some genotypes get lost while others accumulate
hidden as synonyms. Before cutting down an old collection, genotyping is important to verify
that accessions in the new planting are true-to-type (Fernández-Fernández, 2010). Many
collections include cultivars that have been selected, identified and described on basis of
morphological characters. Here, unambiguous genotyping is needed to provide a permanent
identification of accessions (Garkava-Gustavsson et al., 2008). However, it should be kept in
mind that genotyping is complimentary to phenotyping, and that some accessions with
superior horticultural traits or specific cultural-historical value in many cases deserve special
attention.
European apple germplasm
Single sequence repeat (SSR) markers have been the choice of markers for genotyping many
apple cultivar collections (Hokanson et al., 2001; Guarino et al., 2006; Garkava-Gustavsson et
al., 2008; Pereira-Lorenzo et al., 2008; Gasi et al., 2010; van Treuren et al., 2010; Evans et al.,
2011; Gross et al., 2012; Potts et al., 2012; Urrestarazu et al., 2012; Liang et al., 2015; Lassois
et al., 2016). Here, SSR marker data has exposed genetic diversity, identified cultivars and
revealed parentages. These markers have also proven to be a useful tool for gene bank
18. 14
curators in order to reveal mislabelling, synonyms, to identify true-to-type genotypes and
hence prioritize accessions.
Core collections representing maximum genetic diversity and a minimum of genetic
repetitiveness have been recommended to encourage a better and more efficient use of plant
genetic resources (FAO, 1996). Core collections imply a number of benefits since they allow
targeting of available resources and help to focus conservation efforts in facilitating better
utilization of germplasm accessions. Core collections have been established in a number of
apple collections (Liang et al., 2015; Lassois et al., 2016).
Germplasm conservation efforts in Europe have been more or less un-coordinated and
performed at regional or national level (van Treuren et al., 2010; Urrestarazu et al., 2012).
Until recently, the genetic diversity in apple germplasm at a European level has remained
unexposed. Urrestarazu et al. (2016) made the first survey that successfully genotyped more
than 2400 apple accessions from a broad geographical range in Europe. The survey exposed
three genetic groups reflecting geographical origin with a North-East, West and South
European cluster.
Exploring genetic diversity in the collection
It is expected that several genotypes have never been included in the Danish apple collection
nor described in literature. In order to search for such unknown genotypes, 76 accessions from
the well-reputed private nursery Assens Planteskole (Funen, Denmark) were established at the
Pometum in 2009. In order to expose duplicates, identify unknown genotypes and compare
genotypes and phenotypes between the Assens and the Pometum collection a genetic
fingerprinting study was set up. 15 SSR markers were applied to the Danish collection
(n=287), a reference set of cultivars primarily from other European countries (n=86), the
Assens collection (n=76) and a selection of M. sieversii, M. sylvestris and small-fruited,
ornamental cultivars belonging to M. baccata, M. floribunda and M. sieboldii (n=36)
(Manuscript I). The study revealed unambiguous identification of genotypes, pinpointing of
duplicates, revealed several parentages and presented a putative genetic structure. In addition,
flow cytometry allowed us to establish ploidy levels which revealed 22% triploid accessions
in the Danish cultivar collection.
Tracking down the origin of cultivars
Genotyping is useful to expose the parentages, e.g. by revealing common origin such as
parent-offspring or sibling relationships. Knowledge on ancestry and genetic diversity is of
major importance in breeding programmes for selection of genetically distinct breeding
partners. SSR fingerprinting allowed us to study the putative relatedness like parent-offspring
relationships characterized by having 50% shared alleles in the case of diploid individuals.
However, this was more complex for triploid cultivars since most software does not include
analysis of polyploids. This complicated the identification of parent-offspring relations which
are expected to share 1/3 of the alleles with one parent and 2/3 of the alleles with the other
parent which could be a triploid itself or a diploid delivering an unreduced gamete.
19. 15
Measurement of genetic diversity
Genotyping can be done using low-density marker systems such as single sequence repeat
(SSR), amplified fragment length polymorphism (AFLP) or restriction fragment length
polymorphism (RFLP) markers. Progress in molecular genetics has allowed the introduction
of genome-wide marker systems. For this purpose single nucleotide polymorphisms (SNPs)
marker systems such as SNP arrays (Chagné et al., 2012; Bianco et al., 2014; Bianco et al.,
2016) and genotyping-by-sequencing (GBS) (Elshire et al., 2011) have been developed which
allows high-resolution genotyping. SSR-markers however, provide precise predictions of
parent-offspring associations. It has been noted, that SSRs are more informative on single
marker level than SNPs, and that eight SNPs are required to provide the same discriminative
power as provided in one single SSR locus (Ayres, 2005). However, high-throughput
phenotyping techniques will probably replace or complement the use of low-density marker
systems to characterize gene bank resources in the years ahead. High-density marker systems
have also shown powerful results in QTL-mapping by high-resolution linkage mapping or
genome wide association studies (GWAS) in which thousands of markers are needed
(Ingvarsson and Street 2011).
In the present work, genotyping-by-sequencing allowed us to generate 15,802 SNPs and use
them to expose the genetic structure and diversity among 380 accessions (Manuscript IV)
which constituted a subset of the accessions that were used for SSR genotyping. The parental
analysis, duplicates and ploidy levels presented with SSR markers (Manuscript I) were
confirmed by the SNP data (Manuscript IV). PCA plot (Figure 1) presents a putative structure
where ‘Cox’s Orange’ with first degree relatives and the “Pigeon” types each comprice a
cluster separate from the remaning cultivars not belonging to any of the two groups.
Apple breeding programmes
Fruit quality and disease resistance are the major breeding goals in international breeding
programmes (Laurens, 1998; Brown et al., 2003; Sansavini et al., 2004). The genetic starting
material for many modern breeding programmes has been a few successful cultivars and their
derivatives, a strategy implying a high risk of inbreeding depression, loss of genetic diversity
and consequently lower robustness to accomplish e.g. environmental changes (Noiton and
Alspach, 1996). Simultaneously, few successful cultivars have been clonally propagated to a
large extent which has created a highly productive cultivation practice of homogeneous trees
and fruits favourable for the world market, with dramatic loss in orchard genetic diversity
(Urrestarazu et al., 2016). In addition, some elite cultivars such as ‘McIntoch’ and
‘Gravensteiner’ have been vegetatively propagated for centuries and thus, not allowed to
evolve simultaneously with pathogens and climate changes (Myles, 2013). Even though this
practice has obvious benefits for present cultivation and marketing practices it may finally be
an obstacle for breeding efforts.
20. 16
Figure 1. Principal components analysis (PCA) performed on Malus domestica cultivars. Only unique
genotypes included. A: PCA plot made on basis of SSR data from 344 accessions. B: PCA made on
SNP (GBS) data among 282 cultivars.
21. 17
At world level, the highest number of new apple cultivars (280) was released from the
European continent in a 10-year period from 1991. This was followed by releases from North
America, Asia and Oceania, all with more than 80 cultivars during the same period. At
national level, USA, New Zealand, Japan and Russia released the highest number of cultivars
(all>50) in the same period. In Europe, the main cultivar founders from this period are
‘Ariwa’, ‘Braeburn’, ‘Discovery’, ‘Elstar’, ‘Florina’, ‘Fuji’, ‘Gala’, ‘GoldRush’, ‘Golden
Delicious’, ‘Idared’, ‘Pink Lady’, ‘Pinova’, ‘Prima’, ‘Red Delicious’ and ‘Topaz’ (Sansavini
et al., 2004).
Nordic apple breeding
Apple breeding is carried out in three countries in the Nordic Region; Sweden, Norway and
Finland by Balsgaard, University of Agricultural Sciences, Graminor AS and MTT Agrifood
Research, respectively (Nybom, 2012). In Denmark, targeted breeding programs were given
up in the 1950’ies at the Governmental Research Station, Blangstedgaard, Odense (Øydvin,
2010). In recent years in Denmark, efforts have been focused on description and evaluation of
existing apple material at the Pometum. Here, a number of phenotypic traits have been
described in “The apple key” (Toldam-Andersen et al., 2011) in addition to quantification of
fruit flavour volatiles (Varming et al., 2013). A low-cost, low-scientific on-going project,
Æble-oaser is currently being carried out. Here, open pollinated seeds from selected parents
have been distributed to citizens around Denmark who then takes responsibility for growing
and finally evaluating the new genotypes.
Some of the germplasm accessions previously phenotyped at the Pometum were included in a
breeding experiment during the present study. Breeding partners were mainly selected among
genotypes with reported local origin, that are adapted to local climatic conditions, with
superior food qualities and preferably of local-cultural value. Disease resistance was also
considered. To avoid hybridization incompatibility and inbreeding problems, S-RNase alleles
and genetic affiliation among breeding partners were considered. The breeding scheme is
outlined in Table 1. For the early ripening cultivars, ‘Skovfoged’ and ‘Guldborg’ all fruits
unfortunately aborted before fruit ripening. Seeds were gained from three cross combinations.
Seedlings have so far been obtained from one cross, performed in 2014 whereas the seeds
from crosses performed in 2016 have not yet germinated.
Table 1. Apple crossings made at the Pometum (University of Copenhagen, Denmark) in 2014 and
2016.
FEMALE MALE No. pollinations No. fruits No. seedlings
01.05.2014 Ildrød Pigeon (140a) Dronning Louise (33b) 50 -
Dronning Louise (33b) Ildrød Pigeon (140a) 60 15 20
Skovfoged (161a) Dronning Louise (33b) 40 -
13.05.2016 Ritt Bjerregaard (BH) Dronning Louise (33b) 60 10
Dronning Louise (33b) Ritt Bjerregaard (BH) 80 15
14.05.2016 Guldborg (67a) Dronning Louise (33b) 40 -
C.J.H. 12-32 (203b) Discovery (U67) 30 -
22. 18
Maintenance of compatibility
A strict out-breeding mechanism in apple keeps a great diversity and a high level of
heterozygosity over many generations. Inbreeding has been prevented by a gametophytic self-
incompatibility system, which prevents self-fertilization and mating between close relatives. It
is genetically controlled by the S-locus (Kobel et al., 1939) where differences in S-RNase
alleles of pollen and stigma are required for successful fertilization. In natural populations,
pollen bearing a rare S-RNase allele has increased mating chances; whereas pollen bearing a
high-frequency allele has reduced mating chance (Wright, 1939). This keeps a balanced
frequency among alleles and high diversity of S-RNase alleles over the years (De Franceschi
et al., 2012). Modern cultivars that descend from breeding programmes founded on a reduced
number of popular genotypes have a lower diversity in S-RNase alleles compared to old
cultivars originating from unattended pollinations (Broothaerts et al., 2004; Dreesen et al.,
2010). This is not of immediate importance for most commercial orchards where small-fruited
ornamental cultivars frequently are used as pollen donors (Nybom et al., 2008). But it is
crucial to private growers and breeding efforts where incompatibility and finally fruit setting
problems may be faced.
23. 19
Genome-wide approaches in apple
Phenome to genome
Understanding the link between phenotypes and genotypes is a major goal for plant
geneticists and plant breeders today. Traditionally, linkage mapping has been widely used in
annual crops such as barley (Hordeum vulgare L.) where controlled crossings are made to
create a sibling family where segregation of genetic markers among phenotypes has been
studied. However, this approach does not expose the total possible phenotypic variation and
the QTLs that are positioned in larger genomic regions (Myles et al., 2009). In perennial
crops, where several years are required from seedling stage until phenotyping is possible, this
approach is difficult to apply. Indeed, genome-to-phenome association mapping has also been
performed in populations of individuals with unknown relatedness and has been extensively
used in human disease studies (Donnelly, 2008). In a population the approch allows higher
mapping resolution since it builds on recombination events from several generations in the
evolution of the population (Myles et al., 2009). In addition, since the approach does not
study the performance of individuals from controlled crosses and can be applied directly for
mapping in populations without recorded pedigree, it is very useful in a perennial crop. For
accurate predictions of complex traits, however, whole-genome information is required (de
los Campos et al., 2013; Varshney et al., 2014) and fortunately, progress in the field of plant
genomics has allowed whole-genome sequencing of the apple genome (Velasco et al., 2010).
Genome-wide association
The purpose of genome-wide association studies (GWAS) is to find the link between
genotypes and the phenotypic performance. Identification of QTLs for quantitative traits with
a continuous variation is complicated, since the phenotype is the result of the sum of several
small effects caused by several genes plus influence from the environment. For qualitative
traits where the trait falls into different relatively well-defined categories, the GWAS is
generally less complicated. Ideally, a sufficiently high number of markers are used to find
links to all functional alleles. However, even present genome-wide marker systems in apple
such as SNP-chip arrays (Chagné et al., 2012; Bianco et al., 2014; Bianco et al., 2016), RAD-
seq (Sun et al., 2015) and GBS (Gardner et al., 2014) are unlikely to detect all allelic
polymorphisms involved in expression of specific traits. Instead, the assumption is that a
certain number of GWAS markers are in linkage disequilibrium (LD) with the causal allele.
Since LD describes “the non-random association of alleles at different loci” (Flint-Garcia et
al., 2003), LD is decisive for the resolution power of the association study. The closer two
markers are, the stronger is the LD. The decay of LD varies dramatically between species due
to different breeding systems (Flint-Garcia et al., 2003) and knowledge of LD decay in a
particular species is therefore crucial to determine the resolution power of the GWAS.
The relevance of LD in marker-trait association studies is exemplified in Figure 2 for number
of berries in grapevine (Vitis vinifera L.) (Myles et al., 2009). In this example, the casual
(functional) SNP was not genotyped by the GWAS approach. However, the genotyped high
LD SNP is significantly correlated with the causal SNP (p-value=0.037) whereas the
24. 20
genotyped low LD SNP lacks significant association (p-value=0.77). Thus, in the high LD
SNP locus the C allele is correlated with a higher number of berries.
To obtain successful linkage mapping, high-resolution genotyping and accurate and precise
phenotyping are equally important (Pieruschka and Poorter, 2012; Cobb et al., 2013). Current
progress in high-resolution genotyping techniques allows a constant increase in the resolution
power of genotyping techniques for still lower costs. Thanks to standardized genotyping
protocols it is possible to compare results generated between laboratories. However,
phenotype data used for GWAS may, in many cases, imply a number of complications,
especially where data is registered by different people, at different sites, among different
seasons. Such data can be extremely difficult to compare and proper phenotyping therefore
often require replicated records among different sites or seasons which makes phenotyping
slow and costly. Many European apple collections that have been genotyped in recent years
frequently lack evaluation of horticultural traits (Urrestarazu et al., 2016). Standardized
protocols for accurate phenotyping are therefore needed. All in all, they are crucial for
GWAS, for qualified identification of breeding partners in a breeding programme and for
better prioritization gene bank accessions in conservation management.
Figure 2. Marker association for number of berries in grapevine. Genotype Data-box shows the alleles
in two genotyped loci. Low LD SNP (left) are not in significant association to the causal SNP (grey),
whereas High LD SNP (right) are significantly associated to the casual SNP (after Myles et al., 2009).
26. 22
Genome-wide association studies have been made in apple where QTLs, especially for fruit
quality traits, have been explored (e.g. Chagné et al., 2014; Kumar et al., 2015; Sun et al.,
2015; Migicovsky et al., 2016). However, marker-trait associations have been established
using both QTL mapping and GWAS and a number of candidate genes (CGs) have been
proposed (Table 2). Most CGs are suggested because of close proximity to high LD loci. Such
CGs are recognized as positional CGs, whereas cloned genes with a supposed effect on a
particular trait are known as functional CGs (Pflieger et al., 2001).
Genomic selection
In plant breeding, marker-assisted selection (MAS) has been widely used for selection based
on significant linkage between a particular marker and variation for a trait. However, the
technique has been unable to catch loci with minor contributions to the trait. The concept of
genomic selection (GS) uses markers distributed throughout the genome so that all qualitative
trait loci (QTL) ideally are in LD with at least one marker (Goddard and Hayes, 2007).
Therefore, a large number of SNPs revealed by whole-genome sequencing is needed. The GS
starts with a training population which is both genotyped and phenotyped. Derivatives from
the training population form the breeding population which is genotyped but not phenotyped.
From here, new breeding lines are selected. The selection is made on basis of the highest
genomic estimated breeding values (GEBVs) to predict the breeding value in derivative
phenotypes (Desta and Ortiz, 2014).
Genomic breeding
Standard breeding in apple follow three steps as outlined by Kumar et al. (2012). The first
step includes identification of parental lines, creation of controlled pollinations and selection
of superior seedlings. Step two includes multiplication of the selected offspring by grafting
and trialling at various locations. The final step is to test outstanding genotypes in
experimental or commercial orchards. It takes about eight years from the initial cross until
selected superior genotypes can act as breeding partners.
A number of complications in apple breeding such as self-incompatibility and risk of
inbreeding depression can be predicted and, thus, avoided using genomic estimation.
Furthermore, GS allows selection already at seedling stage. Therefore, long-lived perennial
species, such as apple, stand to benefit most from novel genomic breeding approaches
(Gardner et al., 2014). Since only offspring carrying genes-of-interest needs to be grown until
fruiting stage it allows to scale down the number of trees that are kept for final phenotyping
and selection.
Mapping the Danish collection
We made a genome-wide association study using genotypes generated by GBS (Elshire et al.,
2011) by the sequencing service at Cornell University, USA. The method was chosen because
of its low-cost efficiency to produce high-quality SNP data (Gardner et al., 2014). As outlined
by Myles (2013), in high-diversity species such as apples, GBS provides high-quality SNPs
and allows discovery of markers and genotyping in a single step at lower cost compared to
27. 23
competing methods such as SNP arrays. In addition, it has proven useful for genome-wide
mapping in apple (Migicovsky et al., 2016). However, currently available SNP arrays
(Chagné et al., 2012; Bianco et al., 2014; Bianco et al., 2016) have, on the other hand, a
number of advantages to GBS, such as the potential to yield a higher number of SNPs.
Furthermore, the SNP array might be designed to favour SNPs in the coding sequences, only
allowing SNPs with high minor allele frequencies and may ensure a homogeneous
distribution of markers.
Genotypes were obtained for 380 accessions and yielded 29,494 SNP-markers. Applying
various filters such as filtering for minor allele frequency (MAF), missing data, ploidy,
duplicates etc. (as described in Manuscript IV) we ended up with 15,802 SNPs for 248
accessions. These genotypes were used for GWAS in combination with phenotypic data from
three previous projects performed at the Pometum collection. First, we included data for more
than 60 different fruit- and tree characters recorded in the field at the Pometum. These data
have previously been used for creating “The apple key” (Toldam-Andersen et al., 2011)
which include records such as floral and vegetative characters, harvest season, postharvest
longevity, and external and internal fruit characters. We also performed a GWAS for
individual sugar and acid quantities. Finally, fruit volatile compounds obtained by headspace
Gas Chromatography-Mass Spectrometry (GC-MS) analysis from the previous “YDUN-
juice” project (Martinez Vega, 2012) in which juice samples from 200 Danish cultivars
harvested in 2010 were used. Additional data were generated in 2014 from about 100
supplementary cultivars not included in the “YDUN-juice” project to obtain aroma volatile
data for the full set of cultivars in the Danish collection (further outlined in Manuscript IV).
Biosynthesis of aroma volatiles
Apple aroma is a biochemically and genetically complex trait. Even though more than 350
volatile aroma compounds have been identified in apples (Fuhrmann and Grosch, 2002), a
subset of about 20 chemicals have been listed as “character impact compounds” (Dixon and
Hewett, 2000). Esters are by far the largest group of volatiles responsible for the fresh, fruity
apple flavour, though also alcohols, in addition to aldehydes, ketones, terpenes and
polypropanoids contributes to the aroma (Dimick and Hoskin, 1983).
Biosynthesis of important apple aroma volatiles proceeds throughout fruit ripening and
undertakes at least four pathways. The mevalonate pathway leads to α-farnesene synthesis
whereas estragole is synthesised via the phenylpropanoid pathway (Schaffer et al., 2007).
Esters are synthesised via the fatty acid pathway or the Isoleucine pathway (Figure 3). Ester
synthesis via the fatty acid pathway is initiated by degradation of the fatty acids to form
straight chain esters (Rowan et al., 1999). Branched chain esters, on the other hand, are
synthesized from Isoleucine (Rowan et al., 1996) which is produced from amino acids,
namely threonine which again derives from aspartate (Azevedo et al., 1997). Aldehydes are
produced from both the fatty acid and Isoleucine degradation pathways and are reduced to
alcohols via alcohol dehydrogenase. The final step in ester synthesis is catalysed by alcohol
28. 24
acyl transferases (AATs) (D'Auria, 2006). Esters can, however, again be degraded into
alcohols and acids by carboxylesterases (Souleyre et al., 2011).
Figure 3. Simplified schematic overview of the two pathways for ester biosynthesis. The fatty acid
pathway yields straight chain esters whereas the Isoleuine biosynthesis pathway contributes to
branched chain esters. Selected enzymes are shown above arrows (after Schaffer et al. 2007).
Main aroma volatiles
The two major volatiles among various apple cultivars are hexyl acetate and butyl acetate
according to Karlsen et al. (1999). In this study the major volatiles contributing to the apple
flavour in a group of commercial cultivars were found to be butyl acetate, 2-methylbutyl
acetate, hexyl acetate, propyl acetate, ethyl acetate, ethyl butanoate, 1-butanol, ethanol and α-
farnesene. In the two cultivars ‘Cox’s Orange’ and ‘Elstar’, the first three of these volatiles
were in addition to (E)-β-damascenone, cis-3-hexenal and cis-2-nonenal reported as character
impact compounds (Fuhrmann and Grosch, 2002). While hexyl-, butyl-, propyl-, 2-
methylbutyl acetate and (E)-β-damascenone are characterized as contributors to fruity/sweet
and pungent flavour, ethyl acetate, ethanol, ethyl butanoate and 1-butanol are recognized as
pineapple and burning taste. Finally, α-farnesene, cis-3-hexenal and cis-2-nonenal has a
green/grassy or cucumber flavour.
Fruit volatile GWAS
Significant SNP-trait association for apple aroma volatiles were found on all 17 chromosomes
(Manuscript IV). In total, we found 797 significant SNP-trait associations for the 49 aroma
volatiles. The major clusters of densely located SNPs associated to multiple volatiles were
found on chromosome 2, 5, 10, 15 and 17. Kumar et al. (2015) found significant association
to multiple esters on chromosome 2, 15, 16 and 17. Dunemann et al. (2009) on the other hand,
found QTLs for aroma volatiles on 12 of the 17 chromosomes but located the major QTLs on
chromosome 2, 3 and 9. Due to frequent reports of significant marker-trait association for
ester biosynthesis on chromosome 2 (Table 2), the top end of chromosome 2 has been
suggested to be a “crucial hot spot” (Kumar et al. 2015) for potential candidate genes
involved in aroma volatile biosynthesis.
29. 25
We identified several SNPs were significantly associated with multiple volatile esters
(Manuscript IV, Supplementary File 4). A number of individual SNPs have marker-trait
association to at least five esters. These were mainly methyl esters and heptanal. In the far end
of chromosome 15 we found a cluster of SNPs associated to multiple volatiles.
We found no significant associations for the two most dominant esters, hexyl acetate and
butyl acetate, which is in accordance with previous reports (Dunemann et al., 2009; Kumar et
al., 2015). However, for both esters we found a dense cluster of non-significant SNPs on
chromosome 2 with the SNP, chr2:1927844 situated 22 Kb from the gene MpAAT1
(MDP0000214714) (-log10(p-value)=3.2 and 3.4, respectively for the two esters). This gene
is involved in biosynthesis of alcohol acyl transferase that catalyses the final step in ester
synthesis and has been suggested as an important candidate gene involved in ester
biosynthesis (Zini et al., 2005; Dunemann et al., 2009; Dunemann et al., 2012; Ulrich and
Dunemann, 2012; Costa et al., 2013; Souleyre et al., 2014; Kumar et al., 2015). We also
found significant SNPs for other acetate esters (methyl acetate, pentyl acetate and propyl
acetate) on chromosome 2, even though located relatively far (>2Mb) from the MpAAT1.
Sugar and acid content
Sweetness and acidity are important parameters for fruit quality and consumer acceptability of
apples (Harker et al., 2003). In photosynthesis, sucrose and sorbitol are produced in source
organs and, via the phloem, translocated to sink organs such as fruits (Li et al., 2012). Here,
they can be synthesised into fructose, glucose, malic acid and starch. Among apple cultivars,
fructose has been found to be the predominant sugar while malic acid was the dominating
organic acid (Wu et al., 2007; Nour et al., 2010). After fruit harvest, glucose content increases
while sucrose content declines, mainly due to sucrose degradation into fructose and glucose
during ripening (Fuleki et al., 1994) catalysed by neutral invertase (Zhu et al., 2013). We
found significant marker-trait associations for the trait sucrose to total sugar ratio (Manuscript
IV) which is in accordance to previous reports (Guan et al., 2015). This led us to suggest a
vacuolar invertase as candidate gene for the trait. Further studies should confirm this by
looking at gene expression and allele polymorphism among cultivars with varying sucrose
content. This could be revealed by performing biochemical assays where enzyme activities for
different allelic versions of the enzyme are studied. It could also be relevant to look for
potential differences in invertase activity along different pH values since different pH optima
varies between extracellular and vacuolar invertases (Goetz and Roitsch, 1999). Probably the
majority of the sugar is stored in the vacuole which is slightly acidic.
Malic acid is the predominant acid in apple and has major impact on the acidity of apples. The
content of malic acid is determined by malic acid transporter gene located on chromosome 16
(Khan et al., 2013). Unfortunately, however, we found no significant associations for this
trait. Neither was significant marker-trait associations found for the total amount of soluble
sugars, °Brix, which has been reported to be the best objective measure of perception of
sweetness (Harker et al., 2003).
30. 26
General discussion
The first aim of the study was to explore the genetic resources in the Danish apple gene bank
collection. This was done using SSR markers and genome-wide SNP data. In addition, we
studied ploidy levels and the S-RNase allele loci. The second aim was to link SNP data with
existing phenotype data for various fruit quality traits in a GWAS.
The Danish apple collection
Genetic diversity and cultivar parentages were explored in the Pometum collection, the
private Assens Planteskole collection and in the reference collection of European cultivars.
We found no genetic structure in this material (Manuscript I) which is similar to the findings
in Swedish and Finnish collections (Garkava-Gustavsson et al., 2008; Garkava-Gustavsson et
al., 2013). Nevertheless, significant genetic structures have been found in a number of other
European collections (Urrestarazu et al., 2012; Liang et al., 2015; Lassois et al., 2016). The
Danish collection does not form a distinct genetic cluster apart from the reference collection,
probably due to a high number of accessions in admixes between the two collections. It
probably also reflects the fact, that the reference collection was selected to reflect the cultivars
that have been most commonly grown in Denmark during the previous century according to
historical data (e.g. Pedersen, 1950). However, the reference collection might not be
genetically representative for the European germplasm diversity.
Previous exploration of the apple genetic diversity at the European level was performed
across 14 apple collections using 16 SSR markers (Urrestarazu et al., 2016). This was
possible due to a standard set of SSR markers recommended to access apple diversity by the
ECPGR Malus/Pyrus working group (Evans et al., 2007). These markers were also used in
the present study making it possible to compare the present data with a much larger European
cultivar assemble. A core set of 96 accessions (kindly provided by Dr. C.-E. Durel) that act as
standards for allele sixes among genotype data obtained in different laboratories, were also
genotyped in the present study. Unfortunately, PCR did not yield sufficient amplification for
these accessions and this core set has so far not been included in further analysis.
Selected sports or synonyms?
In the Danish apple gene bank collection, 10% of the accessions were found to be duplicate
genotypes on basis of the SSR markers (Manuscript I). This finding was confirmed exploring
15,802 SNPs. The set of SSR markers were highly polymorphic (17 – 30 alleles per loci) and
have previous proven powerful for discrepancy at cultivar level among a high number of
cultivars (e.g. Urrestarazu et al., 2016).
Subclones that originate from spontaneous sports were not distinguishable by the markers.
This was also expected since neutral marker systems such as SSR markers are highly unlikely
to expose single SNP polymorphisms in a casual gene that may cause e.g. skin colour
differences. However, differences in skin colour are also likely to descend from epigenetic
changes. Thus, it has been shown that differences in methylation levels in the MYB10
31. 27
promotor influences anthocyanin levels and skin colour (Telias et al., 2011; Peng and
Moriguchi, 2013).
Figure 4. Five distinct Malus domestica ‘Gravensteiner’ colour sports. Artist: Ellen Backe.
Distinctive skin colour subclones represent extremely high degree of genetic repetitiveness.
Therefore, their contribution to the overall genetic diversity is extremely limited. In a number
of cases, however, it might be justified to maintain a reasonable number of subclones in the
collection. Several distinct ‘Gravensteiner’ colour sports (Figure 4), for example, reflects a
long history of cultivation in Denmark and has cultural-historical value. For a number of
subclones the accession name obviously indicates that the subclone has been selected on
purpose such as ‘Rød Guldborg’ and ‘Filippa Harritslev’ due to observed phenotypic
difference from ‘Guldborg’ and ‘Filippa’, respectively. These are highlighted with red colour
on Online resource 4 of Manuscript I, and there might be reasons to keep these subclones in
the collection.
However, in other cases there are reasons to assume that no differences exist between
particular gene bank accessions. Examples are ‘Hillestedæble’ and ‘Hillerslevæble’, where
‘Hillestedæble’ is described in the literature (Matthiessen, 1913) and ‘Hillerslevæble’
obviously descends from a mis-spelling error of ‘Hillestedæble’. In other cases a cultivar has
been moved across the national border and has received a local name. An example is
‘Hornbækæble’ that is a synonym of ‘Beauty of Kent’, which is in accordance with the fact
that synonyms for ‘Beauty of Kent’ exist on a number of European languages (Pedersen,
1950). Such examples exist among gene banks where local synonyms or name translations
have resulted in synonym accessions (Urrestarazu et al., 2016). In other cases, the identity of
an apple tree has been lost and the tree has received a new name. This is most likely the case
32. 28
for ‘Høje Taastrupæble’ that is a synonym of ‘Rød Ananas’. Yet, in other cases the
nomenclatural inconsistency between two cultivar names has long been sorted out such as for
‘Broholm’ and ‘Broholm Rosenæble’ (Pedersen, 1950). Finally, the two synonym accessions
‘Prinsesse Benedicte’ and ‘Prinsesse Anne Marie’ were belived to be invividual selections
from a breeding programme in Beder, Aarhus, Denmark. Both cultivar names have been
reconized since the first half part of the 20th
century; however, it is unknown whether one of
two original genotypes has been dublicated in the gene bank, or whether the two cultivar
names originally was given to the same genotype.
Other Malus accessions
We explored 75 accessions from the private nursery collection, Assens in order to search for
unknown, unique genotypes. We found 7 duplicates within the collection and 15 accessions
were identical to existing accessions in the Pometum collection. The Assens collection holds a
higher proportion of unique alleles (19% of genotypes) than the Pometum (7% of genotypes)
(Manuscript I). It suggests that the collection holds a valuable variation and that at least some
of its 52 unique genotypes should be considered in future conservation strategies.
Horticultural traits, passport information etc. should be considered together with genetic data
in order to select accessions that eventually may be included in the Pometum collection.
In the present study, the cultivars represent a nigh number of cooking apples with some
dessert apples in between including several genotypes in admix. Selection has favoured
cultivars adapted specifically to North European costal climate conditions in addition to
various local preferences. This has resulted in a cultivar ensemble dominated by juicy and tart
flavoured cooking apples with relatively long postharvest storability for a long winter period
(Pedersen, 1950; Toldam-Andersen et al., 2011). It might be that several North European
cultivars have experienced a weak domestication process opposing many New World
cultivars, where there has been a strong selection preference for red apples with sweet flavour
(Migicovsky et al., 2016). This could explain why the Malus domestica cultivars examined
here cluster together with M. sieversii based on SSR data (Manuscript I). However, they are
separated on PCA based on SNP data (Manuscript IV). This is, on the other hand, in
accordance with previous studies that have shown genetic distinction been between M.
domestica and M. sieversii (Salvi et al., 2014; Migicovsky et al., 2016). Nevertheless, in these
studies the included M. domestica cultivars have mainly consisted of sweet dessert apples that
may have been selectied to larger extent that the present cultivars. It has been proposed for
grapevine cultivars, that some cultivars are no more than few generations from their wild
ancestor (Myles et al., 2011). The same may well apply for a number of apple cultivars, since
they have been kept genetically stable through clonally propagation for many years. It finally
suggests that M. sieversii being a close affiliate to the cultivars is a valuable breeding
resource.
Maintenance of compatibility
We developed a new protocol for genotyping S-RNase alleles and exposed 25 different S-
RNase alleles. This is a higher number of alleles than has been exposed by previous studies. It
33. 29
probably reflects the use of general and multiplexed primers in combination with restriction
enzymes which allowed revealing sequence variants in otherwise quite similar S-alleles.
Previous studies in which only allele specific primers for the most common alleles have been
applied might therefore have failed to expose a number of rare alleles. The diversity of the S-
RNase alleles in the Pometum collection should be conserved by future conservation
management strategies. It should also be implemented in breeding programmes since Dreesen
et al. (2010) reported a lower diversity in S-RNase alleles among ‘new’ cultivars originating
from targeted breeding programmes compared to the higher diversity found in ‘traditional’
local cultivars that have originated as chance seedlings.
Danish cultivars in breeding
Only a minority of the Danish cultivars have given origin to successful commercial cultivars.
A central cultivar in Nordic apple breeding is ‘Ingrid Marie’. From targeted breeding, ‘Ingrid
Marie’ and ‘Filippa’ is, to our knowledge, the only Danish cultivars that have given origin to
commercially successful cultivars. They are both parents to ‘Aroma’ whereas ‘Ingrid Marie’
is parent to ‘Elstar’. Even though a number of Danish cultivars have been used as founders in
breeding programmes, the resulting genotypes have never been commercially successful.
Examples are ‘Signe Tillisch’ that is parent to ‘Prinsesse Margrethe’ and ‘Prinsesse
Benedikte’, ‘Pigeon Almindelig’ that is parent to ‘C. J. Hansen 603’ and ‘C. J. Hansen 1017’,
and ‘Filippa’ that is parent to ‘Blangstedgaard nr. 156’. Among cultivars that originate from
random, unattended pollination events, ‘Ingrid Marie’, ‘Ildrød Pigeon’ and ‘Gravensteiner’
are the only cultivars grown in major commercial orchards in Denmark today (DST, 2012).
Breeding for new cultivars
There is a need for new superior cultivars adapted to local climate conditions in the Nordic
Region. Here, parental lines should be selected among genotypes that have proven suitable for
local climate conditions. Breeding partners should have distinctive affiliation. Specific
cultivar groups (Figure 1) such as ‘Cox’s Orange’ derivatives and “Pigeon” apples are both
recognized by their characteristic aroma. The “Pigeon” apples with relatively small fruits and
distinct taste might be useful for specific breeding goals.
For selection of genotypes with superior aroma qualities using MAS, it is needed to
understand the contribution of each flavour volatile to the overall aroma perception. It could
be revealed combining quantitative aroma volatile data with sensory flavour evaluations by a
trained human sensory panel. Since it has been shown that volatile composition varies
between whole unpeeled fruits and apple juice (Fuhrmann and Grosch, 2002), future studies
might also perform aroma volatile analysis on whole fruits. This should be combined with the
general flavour perception of whole fruits and contribute to better understanding of aroma
volatile composition in dessert apples.
Many genotypes may contain specific traits that can be useful for particular breeding aims.
However, the vast majority also contain a high number of undesirable traits, such as
susceptibility to diseases, low yield caused by biennial bearing or vigorous vegetative growth.
34. 30
Here, careful selection during a number of generations is required to facilitate the transfer of
desired traits. No doubt that ‘Gravensteiner’ has been an important cultivar in Denmark.
However, it low pollen fertility (Florin, 1926; Kvaale, 1926), low ability to produce seeds and
low-vitality seedlings (Dahl and Johansson, 1924) and apparently has no offspring in the
Danish collection (Manuscript I). Its low fertility makes ‘Gravensteiner’ unsuitable for
breeding purposes.
Application of GWAS results
Results from the GWAS performed here have shown a number of significant SNP-trait
associations (Manuscript IV). These SNP markers can be used directly for MAS. However, in
order to reveal a causal gene, the candidate gene should be sequenced in a number of cultivars
to look for variations in encoded proteins or their expression using qPCR. Allelic variants of
the same protein could then be further studied by enzyme assays to look for different allele
activities. Afterwards, editing or overexpression of the particular gene could prove the action
of specific alleles. Then, if a generally superior genotype contains a single undesired trait
controlled by a single QTL, the causing allele can then be cloned and transferred between
genotypes. Another perspective is gene editing techniques such as CRISPR-Cas9 (Doudna
and Charpentier, 2014) that have proven able to edit single traits in apple (Nishitani et al.,
2016). If harvest time in apple is controlled by the suggested NAC transcription factor
(Manuscript IV), the ripening time can be modified just by editing this single gene. This
would be useful for breeding of cultivars for the Nordic Region, where relatively early
ripening genotypes are needed for the short growth season. The candidate gene may also have
impact on postharvest storability and the tendency of early ripening cultivars to overripe
rapidly after harvest.
Resolution of the GWAS
Efforts were done in order to sample apples at their optimal ripening time for the aroma
volatile analysis (Manuscript IV). However, the aroma composition changes rapidly during
ripening. It makes it difficult to get well-defined cultivar aroma profiles since it depends on
the exact ripening stage at which the aroma was measured. The exact time at which the apples
were harvested can therefore have major impact on the GWAS. Another highly critical point,
is that the aroma volatile data used for GWAS, was a combination of data from two seasons
(2010 and 2014). This was due to the fact, that the data used here mainly were existing gene
bank records. For higher GWAS resolution, systematic records in a large sample set from
homogeneous plantings recorded during one season including replicates is ideally needed.
Gene banks in a non-static world
A growing interest for old, local cultivars has been seen during the recent years. Focus has
been on processed apple products such as juice, cider and wine in addition to dessert apple
cultivars for private gardens and local small-scale production. Following this, increased
interest on cultivar-specific fruit qualities and historical origin has been observed. Also, the
recent trend ‘New Nordic Diet’ (Mithril et al., 2012) has spread new light on utilizing local
cultivars and landraces. Indeed, public dissemination events such as ‘open door days’ are of
35. 31
upmost importance to maintain a continuous public attention and general awareness on the
importance of plant genetic resources.
Gene bank accessions are preserved for future generations. Nevertheless, in a dynamic world
with eternal shifts in costumer and society needs, gene banks should keep a proper balance
between traditions and renewal. Continuous germplasm exploration should reduce
repetitiveness and at the same time maximize genetic diversity. In the years to come, further
description and evaluation of apple germplasm should facilitate a better accessibility and
utilization.
36. 32
Conclusions & perspectives
The Danish apple gene bank collection contains many unique accessions adapted to local
growth conditions. Several cultivars are of cultural-historical value. In addition, the collection
holds a large genetic variation and traits that might be useful to reach future breeding goals.
The genetic fingerprints are useful for unambiguous identification of accessions. The results
can be used to prioritize accessions and establish a core collection, where phenotypic
characterization and conservation efforts are focused. The collection holds a high diversity in
S-RNase alleles including several alleles that are rare among commercial cultivars. The
findings suggest some minor future adjustments of the collection. Synonym accessions could
be excluded and replaced with a number of unique samples from the private nursery
collection Assens for better utilization of management resources. In addition, a reasonable
number of distinct colour sports should be kept in the collection.
The collection does not form a separate cluster from the reference collection of European
cultivars studied here. However, this should be further explored including a large ensemble of
cultivars from a broad European range in order to shed more light on the way the cultivars
have originated and dispersed. It would probably also pinpoint unidentified duplicates among
collections and shed more light on general genetic similarity. We have suggested a number of
parent-offspring relations which have confirmed some previously assumed parentages but
also revealed many previously unknown parentages. The parentages should be further
explored using high-density SNP data. Pedigree reconstruction is of historical interest and
important knowledge in breeding programmes where breeding partners without close
affiliation are needed.
We identified QTL-flanking markers for a number of important fruit quality traits. These can
be used directly for MAS. In addition, future studies are needed to reveal the effect of
candidate genes. Especially the role of vacuolar invertase on sucrose content (Manuscript IV)
should be of major interest. Also the candidate genes for harvest date and a number of fruit
flavour volatiles should receive further attention. Another relevant study would be to perform
GWAS for resistance to fruit tree canker, Neonectria ditissima Bres. from evaluation of
cultivars’ susceptibility after controlled virulence tests using an inoculation protocol
(Ghasemkhani et al., 2015).
It is a major benefit for GWAS that SNP data, once generated, can be used year-after-year
combined with new phenotype records. Systematic phenotype records should therefore be
documented over several seasons from replicated plantings for high-resolution GWAS. In-
depth characterization using standard protocols would be extremely helpful here; also in order
to pinpoint accessions with superior traits needed to breed for high-quality cultivars for the
Nordic Region.
37. 33
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Manuscript I
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