Molecular basis behind flower colour
determination in lily cultivars
Comparative study between oriental
hybrids and Lilium longiflorum
MSc Thesis
Katarzyna Wolinska
Registration number:
930612970170
MSc Plant Sciences
Supervisors
Nur Fatihah Binti Hasan Nudin
Dr. Frans Krens
Dr. Jan Schaart
Background of the cover page is a
modified picture from Nur Fatihah
Molecular basis behind flower colour
determination in lily cultivars
Comparative study between oriental
hybrids and Lilium longiflorum
MSc Thesis
Katarzyna Wolinska
Registration number:
930612970170
MSc Plant Sciences
Supervisors
Nur Fatihah Binti Hasan Nudin
Dr. Frans Krens
Dr. Jan Schaart
Abstract ................................................................................................................................................... 8
1
INTRODUCTION................................................................................................................................ 9
2
LITERATURE STUDY OF THE CANDIDATE GENES ............................................................................ 11
2.1
Structural genes ..................................................................................................................... 11
2.1.1
Chalcone synthase a and b (CHSa & CHSb) .................................................................... 11
2.1.2
Chalcone isomerase a and b (CHIa & CHIb) ................................................................... 11
2.1.3
Flavanone 3-hydroxylase (F3H) ...................................................................................... 11
2.1.4
Flavonoid 3’-hydroxylase (F3'H) ..................................................................................... 12
2.1.5
Dihydroflavonol 4-reductase (DFR) ................................................................................ 12
2.1.6
Anthocyanidin synthase (ANS) ....................................................................................... 12
2.1.7
Flavonol synthase (FLS) .................................................................................................. 12
2.1.8
Flavonoid 3’-5’- hydroxylase (F3’5’H)............................................................................. 12
2.1.9
UDP-glucose:anthocyanidin 3-glucosyltransferase (3GT) .............................................. 13
2.1.10
Other transferases ......................................................................................................... 13
2.2
Transcription factors .............................................................................................................. 13
2.2.1
Basic helix–loop–helix 2 (bHLH2) ................................................................................... 13
2.2.2
MYB domain protein 15 (MYB15) .................................................................................. 13
2.2.3
MYB domain protein 12 (MYB12) .................................................................................. 14
3
HYPOTHESES .................................................................................................................................. 15
4
MATERIALS & METHODS................................................................................................................ 16
4.1
Materials ................................................................................................................................ 16
4.1.1
Cultivars ......................................................................................................................... 16
4.1.2
Flower stages ................................................................................................................. 17
4.1.3
Genes ............................................................................................................................. 17
4.2
Methods................................................................................................................................. 17
4.2.1
Flower buds sampling .................................................................................................... 17
4.2.2
Grinding ......................................................................................................................... 17
4.2.3
gDNA isolation ............................................................................................................... 17
4.2.4
RNA isolation.................................................................................................................. 18
4.2.5
DNase treatment and cDNA synthesis ........................................................................... 18
4.2.6
Primers design ............................................................................................................... 18
4.2.7
Gene expression analysis by RT-qPCR ............................................................................ 18
4.2.8
Statistical analysis of relative gene expression .............................................................. 19
4.2.9
Sequencing .................................................................................................................... 19
4.2.10
5
RESULTS ......................................................................................................................................... 20
5.1
Evaluation of RNA isolation protocol ..................................................................................... 20
5.2
Genes presence ..................................................................................................................... 21
5.3
Sequence analysis .................................................................................................................. 23
5.3.1
CHSa ............................................................................................................................... 23
5.3.2
CHSb .............................................................................................................................. 23
5.3.3
CHIa................................................................................................................................ 24
5.3.4
CHIb ............................................................................................................................... 27
5.3.5
F3H................................................................................................................................. 28
5.3.6
F3’H................................................................................................................................ 29
5.3.7
DFR................................................................................................................................. 29
5.3.8
ANS ................................................................................................................................ 30
5.3.9
bHLH2 ............................................................................................................................ 32
5.3.10
MYB12............................................................................................................................ 33
5.3.11
MYB15............................................................................................................................ 34
5.4
Gene expression .................................................................................................................... 34
5.4.1
Reference gene evaluation ............................................................................................ 34
5.4.2
Primer dimers ................................................................................................................ 35
5.4.3
Relative Gene Expression ............................................................................................... 35
5.4.3.1
Structural genes ......................................................................................................... 36
5.4.3.1.1
CHSa &CHSb ........................................................................................................ 36
5.4.3.1.2
CHIa & CHIb ......................................................................................................... 36
5.4.3.1.3
F3H ...................................................................................................................... 36
5.4.3.1.4
DFR ...................................................................................................................... 36
5.4.3.1.5
ANS ...................................................................................................................... 36
5.4.3.2
6
Sequence alignment and structure predictions ............................................................. 19
Transcription factors .................................................................................................. 36
5.4.3.2.1
bHLH2 .................................................................................................................. 36
5.4.3.2.2
MYB15 ................................................................................................................. 37
5.4.3.2.3
MYB12 ................................................................................................................. 37
DISCUSSION ................................................................................................................................... 46
6.1
Sequences and gene expression ............................................................................................ 46
6.1.1
Stability of the reference gene....................................................................................... 46
6.1.2
CHSa and CHSb .............................................................................................................. 46
6.1.3
CHIa and CHIb ................................................................................................................ 47
6.1.4
F3H................................................................................................................................. 47
6.1.5
DFR................................................................................................................................. 48
6.1.6
ANS ................................................................................................................................ 49
6.1.7
bHLH2 and MYB15 ......................................................................................................... 49
6.1.8
MYB12............................................................................................................................ 50
6.2
Difference between ‘Gran Tourismo’ and ‘Perth’ .................................................................. 50
6.3
Why is ‘Rialto’ white .............................................................................................................. 50
6.4
Why is ‘Lincoln’ white ............................................................................................................ 51
7
CONCLUSIONS................................................................................................................................ 52
8
Acknowledgments ......................................................................................................................... 53
9
REFERENCES................................................................................................................................... 54
Appendix I CTAB mini RNA-isolation protocol........................................................................................ 57
Appendix II DNase treatment and cDNA synthesis ................................................................................ 58
Appendix III Primer list ........................................................................................................................... 59
Appendix IV qPCR and Phusion protocols .............................................................................................. 61
Appendix V Microspin™ G-50 Columns protocol ................................................................................... 62
Appendix VI Protein sequence alignment .............................................................................................. 63
Appendix VII Polymorphic SNPs in CHIa gene ........................................................................................ 68
Appendix VIII „Blue Experiment” ........................................................................................................... 69
Methodology ..................................................................................................................................... 69
Appendix IX TOPO® Protocol ................................................................................................................. 70
Appendix XDigestion protocol ............................................................................................................... 71
Appendix XI Ligation protocol ................................................................................................................ 73
ABSTRACT
Lilies are one of the most important ornamental crop species in the world. Their most characteristic
feature is colour and on it this study was focused. White lilies from the Longiflorum group play an
important role on the market and yet the genetics behind their colour is mostly unknown. This study
was focused on finding a reason why lilies are white, especially in the Longiflorum group, but also in
the Oriental hybrids group. Additional aim of this study was to get more insight into the anthocyanin
biosynthesis pathway and the genes involved. Two cultivars originating from Oriental lilies– dark red
‘Gran Tourismo’ and pink ‘Perth’ were used for a comparison with white-coloured cultivars, one
belonging to the Orientals (‘Rialto’) and one Longiflorum (‘Lincoln’). Gene expression of most of the
structural genes from the anthocyanin pathway (CHSa, CHSb, CHIa, CHIb, F3H, DFR and ANS) and three
transcription factors (bHLH2, MYB15 ad MYB12) was determined in five flowering stages. Separately
the sequence analysis of the same genes were performed. Results of gene expression and sequencing
lead to the following conclusions. The white colouration of ‘Lincoln’ is most probably caused by lack of
two structural genes in the genome, F3’H and DFR, but lack of bHLH2 transcription factor can also
have an influence. In ‘Rialto’, the most probable cause is a mutation in the DFR gene which influences
its 3D structure and/or a mutation in MYB12 as found by Suzuki et al. (2015). Another important
finding is that bHLH2 does not seem to be essential for induction of anthocyanin genes, as it was
hardly expressed in the red cultivar ‘Gran Tourismo’.
Keywords: Lilium, Oriental hybrids, Lilium longiflorum, flower colour, anthocyanins, gene expression,
CHS, CHI, F3H, F3’H, DFR, ANS , bHLH2, MYB15, MYB12,.
1
INTRODUCTION
Colouration of flowers with pigments has a role in attracting pollinators which are needed for pollen
dispersal and fertilisation, but pigments, such as anthocyanins, also protect the plant from visible and
UV light (Tanaka et al. 2008). Nowadays, the flower colour is also important for customers looking for
flowers fitting their wishes. According to Holland Bulb Market (http://www.hollandbulbmarket.nl/) lily
bulbs account for 25% of the total bulb export of The Netherlands.
Anthocyanins are one of the three main classes of pigments, next to carotenoids and betalains (Zhao
and Tao 2015) and they are responsible for an orange, pink, red and blue colour (Norbaek and Kondo
1999; Davies et al. 2003). Anthocyanins belong to the class of flavonoids, plants secondarymetabolites with the basic chemical structure of C6-C3-C6. They are derived from the unstable
anthocyanidins (which consist of six major groups responsible for a wide range of colours varying from
orange to blue: pelargonidin, petunidin, malvidin, peonidin, delphinidin and cyanidin) to which methyl,
acyl or glucosyl groups are attached by methyl- acyl- or glucosyltransferase. Generally, the more
hydroxyl groups the compound has, the bluer its colour (Tanaka et al. 2008). Other factors influencing
the colour of the pigments are pH of the surrounding area, presence of metal ions and co-pigments
such as flavones and flavonols (Brugliera et al. 2013). Anthocyanins are stored in the vacuoles (Tanaka
et al. 2008) and polar side chains which are often attached to the main chain of anthocyanins are
responsible for their water-soluble properties (Schoefs 2004).
The flavonoid biosynthesis pathway is extensively studied and well characterised (Davies et al. 2003).
Several genes have been discovered, which are involved in a biosynthesis pathway of pink/red
anthocyanins (Figure 1). If the expression of only one of those genes or their transcription factors is
disrupted in flowers, the petals (or tepals in case of lily) will not accumulate anthocyanins and in result
will have a white phenotype (Yamagishi et al., 2010; Yamagishi 2011; Suzuki et al. 2015).
One of the important ornamental flower crops is lily. According to Comber (1949) lilies are divided into
seven groups based on their phenotype: Martagon, Pseudolirium, Lilium (Liriotypus), Archelirion,
Sinomartagon, Leucolirion and Daurolirion (Comber 1949; Lim and Tuyl 2007). According to the Royal
Horticultural Society they are divided into nine horticultural divisions, mostly focused on hybrid
division (Table 1) (The Royal Horticultural Society 2014). Lilies belonging to the Longiflorum group
(division V from Royal Horticultural Society) are white-coloured. Up to now there are no existing
Lilium longiflorum (L. longiflorum) cultivars with a different flower colour than white (The Royal
Horticultural Society 2014). The main purpose of this study is to find out the reason of the lack of
anthocyanin accumulation in L. longiflorum flowers. For that an Oriental lily group will be used as a
reference. Oriental lilies are hybrids obtained by crosses between Lilium speciosum (L. speciosum),
Lilium auratum (L. auratum), Lilium japonicum, Lilium nobilissimum, Lilium parkmanii and Lilium
rubellum. In Oriental lily group, both coloured as well as white cultivars are present (The Royal
Horticultural Society 2014).
Next to the main experiment, another small project was performed involving blue flowers of
Delphinium. The goal of this project was to evaluate the possibility of changing the colour of lilies more
towards blue. F3’5’H and DFR were chosen for a transient flower transformation assay as they are
assumed to be the key enzymes in production of the anthocyanidin delphinidin (Brugliera et al. 2013).
9
Figure 1. Anthocyanin biosynthetic pathway (modified, based on Brugliera et al., 2013 and Ferreyra et al., 2012).
Chemical structures were drawn with use of eMolecules® tool (https://www.emolecules.com/). Enzymes
involved in the pathway: PAL-phenylalanine ammonia lyase; C4H-cinnamate 4-hydroxylase; 4CL-4-coumaarate;
CHS-chalcone synthase; CHI-chalcone isomerase; F3'H-flavonoid 3'-hydroxylase; F3'5'H-flavonoid 3'5'hydroxylase; F3H-flavanone 3-hydroxylase; FLS-flavonol synthase; DFR-dihydroflavonol 4-reductase; ANSanthocyanidin synthase; 3GT-UDP-glucose:anthocyanidin 3-glucosyltransferase.
Table 1. Lily division. Based on (The Royal Horticultural Society 2014)
Division
I
II
III
IV
V
VI
VII
VIII
IX
Name
Asiatic hybrids
Martagon hybrids
Euro-Caucasian hybrids
American hybrids
Longiflorum lilies
Trumpet and Aurelian hybrids
Oriental hybrids
Other hybrids
Species and cultivars of species
10
2
LITERATURE STUDY OF THE CANDIDATE GENES
This study is focused on the genes related to the anthocyanin biosynthesis pathway. Below, a short
description of the relevant genes can be found. The lengths of the genes belongs to a genus Lilium
were taken from GenBank NCBI database (http://www.ncbi.nlm.nih.gov/genbank/).
2.1
STRUCTURAL GENES
2.1.1 CHALCONE SYNTHASE a AND b (CHSa & CHSb)
CHSa and CHSb belong to the CHS multigene family. CHS genes in L. speciosum are approximately
1.1kb long. The amount of genes present in this family depends on the plant species. For example
Petunia hybrida carries at least eight (Koes et al. 1989) and morning glory five functional CHS genes
(Yang et al. 2004). In Lilium three CHS genes were characterised (Nakatsuka et al. 2009). CHS genes
catalyse the reaction between 4-coumaroyl-CoA and malonyl-CoA which results in 4,2,4’,6’tetrahyroxychalcone (Brugliera et al. 2013). In Magnolia sprengeri, its expression is higher in red than
white coloured petals (Shi et al. 2014). In Arabidopsis thaliana (A. thaliana) an increase in CHS enzyme
activity due to light stress conditions caused a 15-fold increase in anthocyanin content in the leaves
and stems (Feinbaum and Ausubel 1992). Sun et al. (2015) analysed the expression of FhCHS1 gene
(phylogenetically more related to CHSa than to CHSb gene from Petunia hybrida) in Freesia hybrida.
They found that its expression is much higher in flowers than in other tissues and it is increasing in line
with an increase of anthocyanin accumulation over flower development (Sun et al. 2015).
2.1.2 CHALCONE ISOMERASE a AND b (CHIa & CHIb)
CHI catalyses the isomerization of naringenin chalcone (4,2’,4’,6’-tetrahydroxychalcone) into
naringenin (Brugliera et al. 2013; Sun et al. 2015). Up to this date, several CHI genes were found and
they fall into two catalytic categories, one of which is characteristic for leguminous plants and the
other for non-leguminous plants (Shimada et al. 2003). Two chalcone isomerases are known to be
involved in anthocyanin synthesis in lily flowers, CHIa and CHIb (Suzuki et al. 2015). CHI genes in L.
speciosum were found to be approximately 1kb long. The results obtained by Suzuki et al. (2015) in L.
speciosum indicate that, in contrast to most of the genes from the anthocyanin pathway (including
CHIb), CHIa is not regulated by MYB12 transcription factor (Suzuki et al. 2015). In Arabidopsis two
knock-out mutants of two CHI genes (tt5-2 with a distrupted promoter and tt5-3 with an insertion
within one of the introns) did not accumulate kaempferol or quercetin (Bowerman et al. 2012), which
suggests the anthocyanin pathway is disrupted.
2.1.3 FLAVANONE 3-HYDROXYLASE (F3H)
F3H catalyses a change of pentahydroxyflavanone, eriodictyol and naringenin to dihydromyricetin,
dihydroquercetin and dihydrokaempferol (Brugliera et al. 2013). In Dianthus caryophyllus, F3H is a
single copy gene, and when silenced (with the antisense sequence) the colour of the petals changes
from dark orange to white/creamy. Orange pelargonidin which is the only pelargonidin responsible for
the flower colour in this cultivar (due to lack of F3’H and F3’5’H activity) was present in a very low
amount in the transformed plants. Fragrance of the flowers was also altered due to the F3H silencing
(Zuker et al. 2002). In L. speciosum F3H gene has a length of approximately 1.2kb.
11
2.1.4 FLAVONOID 3’-HYDROXYLASE (F3'H)
F3’H is a ~1.7kb long gene and it encodes an enzyme which can transform several different chemical
compounds. Examples of substrates for F3’H are: kaempferol, dihydrokaempferol naringenin and
apigenin (Hagmann et al. 1983; Ueyama et al. 2002). Overexpression of F3’H causes an increased
accumulation of anthocyanins from the cyanidin group and results in a redder flower phenotype
(Ueyama et al. 2002).
2.1.5 DIHYDROFLAVONOL 4-REDUCTASE (DFR)
DFR catalyses the first step of the anthocyanin biosynthesis from dihydroflavonols (Davies et al. 2003)
and it is approximately 1.4kb long. In Bromheadia finlaysoniana and Cymbidium hybrid, the DFR was
found to be a single copy gene (Neo and Ho 2001). Its expression is higher in red petals of Magnolia
sprengeri than in the white ones (Shi et al. 2014). Lilies with a dysfunctional DFR gene were whitecoloured with yellow anthers. A cross between two lilies with white flowers (one with yellow anthers
and the other with red ones) resulted in progeny with pink flowers. That indicates the recessiveness
of the mutated DFR gene. The parental line with red anthers has white tepals because of a mutation in
a MYB12 transcription factor (this mutation is lacking in white-yellow phenotype (Suzuki et al. 2015)).
Introduction of a DFR gene with a constitutive promoter in Mitchel Petunia (originally white petals due
to a mutation in MYB-like transcription factor and anthocyanin O-methyltransferase) resulted in
anthocyanin accumulation and pink petals (Davies et al. 2003).
2.1.6 ANTHOCYANIDIN SYNTHASE (ANS)
ANS catalyses the first step in obtaining the coloured anthocyanidins, which later can be transformed
by other enzymes into coloured, stable anthocyanins (cyanidin 3-glucoside, pelargonidin 3-glucoside
and delphinidin 3-glucoside).Its length in lilies is approximately 1.4kb. iRNA suppression of ANS in
Torenia hybrid decreased the ANS expression in almost 90% of these plants. In half of all the
transformed plants a loss of colour was observed resulting in white-coloured flowers, while 38%
displayed a paler flower colour (Nakamura et al. 2006).
2.1.7 FLAVONOL SYNTHASE (FLS)
FLS catalyses the biosynthesis of colourless flavonols with dihydroflavonols as substrates. When its
expression in Petunia petals is silenced, flavonol level decreases and accumulation of anthocyanins
occurs resulting in a pink or red phenotype. In Nicotiana tabacum, silencing of the FLS gene caused a
phenotypic change from light pink to red flowers (Holton et al. 1993; Davies et al. 2003)
2.1.8 FLAVONOID 3’-5’- HYDROXYLASE (F3’5’H)
F3’5’H encodes an enzyme which catalyses the transformation of naringenin to
pentahydroxyflavanone (Figure 1). It is one of the steps in the anthocyanin pathway leading to the
synthesis of the compounds responsible for blue colouration (Brugliera et al. 2013). It is not naturally
found in most of the ornamental plants such as lilies or orchids (Sasaki and Nakayama 2015),
nevertheless, after the introduction of an exogenous F3’5’H gene originating from Viola wittrockiana
12
cv. ‘Black Pansy’ into the chrysanthemum genome (another group of ornamental plants) a switch
towards blue flower colour was observed (Brugliera et al. 2013).
2.1.9 UDP-GLUCOSE:ANTHOCYANIDIN 3-GLUCOSYLTRANSFERASE (3GT)
3GT catalyses the transformation of anthocyanidins into stable anthocyanins by attaching a glucosyl
moiety to a substrate. In Iris hollandica, the enzyme encoded by 3GT has a wide substrate range, but
with different specificity, with pelargonidin having the lowest and malvidin the highest specific activity
(Yoshihara et al. 2005). Although not much is known about the cellular localisation of
glucosyltransferases in plants, for now the most convincing theory is that they are mostly placed in
cytoplasm (Ross et al. 2001). There are glucosyltransferases which are found in Golgi apparatus, but
UDP-Glucose was not one of them (Nikolovski et al. 2012).
2.1.10 OTHER TRANSFERASES
Other transferases include glucosyl-, acyl- and rhamnosyl-transferases (Sasaki and Nakayama 2015).
Glucosyltransferases transfer an activated sugar – such as UDP-glucose on their substrates (Vogt and
Jones 2000). Glucosyltransferases are considered to be cytosolic, with only few vacuolar exceptions
(Vogt and Jones 2000). Acylation of the anthocyanins occurs in vacuoles and it increases the pigments
and colour stability (Sasaki and Nakayama 2015). Secondary glucosylation of anthocyanidin 3glucoside is catalysed by acyl-glucose-dependent anthocyanin 5-O-glucosyltransferase and acylglucose-dependent anthocyanin 7-O-glucosyltransferase. The glucosylation occurs at the 5- or 7position respectively (Nishizaki et al. 2013). Acylation in 3- and 5-position gives reddish/purple colour,
while the acylation in 3- and 7-position results in more blue colouration (Sasaki and Nakayama 2015).
2.2
TRANSCRIPTION FACTORS
2.2.1 BASIC HELIX–LOOP–HELIX 2 (bHLH2)
bHLH2 is a transcription factor from Asiatic hybrid lily (Suzuki et al., 2015) homologous to the AN1
gene in Petunia. In Petunia, its interaction with the Petunia homologue of R2R3-MYB transcription
factor regulates the genes of the anthocyanin biosynthesis pathway. Its expression is highly correlated
with the anthocyanin content (Nakatsuka et al. 2009; Suzuki et al. 2015). In Asiatic hybrid lily the
bHLH2 gene was expressed in several different tissues, such as tepals, filaments, leaves or stems. In
Asiatic lily tepals, the expression of bHLH2 was first increasing from stage 1 (bud size 3 – 4cm) till stage
2 or 3 (bud size 4 – 5cm or 5 – 6cm, depending on the cultivar) and then decreasing until the flower
was fully opened (Nakatsuka et al. 2009). The bHLH2 is approximately 2.4kb long.
2.2.2 MYB DOMAIN PROTEIN 15 (MYB15)
MYB15 is a newly discovered transcription factor from Lilium regale regulating the early and late stage
genes of anthocyanin biosynthesis with the length of approximately 0.9kb. After introducing in
tobacco plants it induces anthocyanin accumulation in several tissues (Yamagishi 2016). In A. thaliana
subjected to a salt stress, the expression of MYB15 in leaves increased 16 folds and resulted in the
increased accumulation of anthocyanins (Jiang and Deyholos 2006). Overexpression of MYB15 in A.
thaliana resulted in an increased tolerance to drought and salt stress (Ding et al. 2009).
13
2.2.3 MYB DOMAIN PROTEIN 12 (MYB12)
MYB12 is a transcription factor involved in anthocyanin biosynthesis with the length of approximately
1.1kb. The higher the MYB12 was expressed, the higher expression of several structural genes was
detected (CHSa, CHSb, CHIb, F3H, F3’H, DFR and ANS) (Suzuki et al. 2015). A single nucleotide
substitution can cause a premature stop-codon and make a gene non-functional. In case of MYB12
this mutation leads to a failure to induce the expression of the structural genes (Suzuki et al. 2015).
14
3
HYPOTHESES
The preliminary analysis of the pigment composition in white flowers of ‘Snow Queen’ cultivar (Lilium
longiflorum) revealed a presence of the early metabolites from the anthocyanin synthesis pathway.
Dihydrokaempferol, dihydroquercetin, and quercetin were present, which suggests that the genes
CHS(a and/or b), CHI(a and/or b), F3H and F3'H are present and expressed in the flowers. Several
possibilities explaining the lack of further metabolites are listed below:
1) Lack of the functional genes for the subsequent steps in the pathway.
2) MYB12 is non-functional and it does not induce the expression of the genes in flower stages 3 – 5
(Yamagishi 2010). The transcription factor responsible for activating the whole anthocyanin pathway is
expressed only in the early stages of flower formation and is turned off in the later stage.
3) Based on the findings of Suzuki et al. (2015) it is expected to find a substitution – or another
mutation – in MYB12 and/or a mutation resulting in a pre-mature stop codon in DFR or ANS in the L.
longiflorum cultivar ‘Lincoln’, which causes the genes to be non-functional.
4) In the cultivars with coloured flowers (‘Perth’ and 'Gran Tourismo'), the expression of all the genes
involved in the pathway, is expected to be detectable. For darker-coloured flowers ('Gran Tourismo')
the expression of the genes is expected to be higher than for lighter-coloured flower (‘Perth’).
5) Gene expression pattern in ‘Perth’ is expected to be similar to a ‘Sorbonne’ cultivar, as their tepal
colour is very similar. , with the assumption that the pigments responsible for the tepal colour are the
same for both cultivars as they belong to the same hybrid group.
In order to prove the hypothesis true or false an experiment involving gene expression analysis in
three Oriental lilies (with a colouration of red, pink and white) and one L. longiflorum lily at different
flowering stages was performed. Additionally sequences of the target genes were obtained to prove
their presence/absence in the genome.
For the “blue” experiment, two genes (F3’5’H and DFR) originating from Delphinium 'Delfix Blue'
flowers are expected to alter anthocyanin production in transformed lilies. The expectation is to shift
the anthocyanin production towards delphinidin and the colour to blue.
15
4
4.1
MATERIALS & METHODS
MATERIALS
4.1.1 CULTIVARS
Four different lily cultivars were grown. The representative of L. longiflorum is the cultivar ‘‘Lincoln’’,
on which the main focus is set. The other three are: ‘Rialto’, ‘Perth’ and Gran Tourismo. Those three
cultivars represent the group of Oriental lilies in a range of colours from white to red (Table 2; Figure
2). For blue experiment one cultivar of commercially available Delphinium 'Delfix Blue' was bought in
June 2015, frozen in liquid nitrogen and stored in the -80oC freezer until the beginning of the
experiment.
Table 2. Lily cultivars used in this study
Cultivar
‘Lincoln’
‘Rialto’
‘Perth’
'Gran Tourismo'
Group
Longiflorum
Oriental
Oriental
Oriental
Tepal colour
White
White
Bright pink
Dark red
Figure 2. Lily cultivars with fully opened flowers. Top from left to right ‘Lincoln’ and ‘Rialto’, bottom from left to
right ‘Perth’ and 'Gran Tourismo' (pictures taken by Nur Fatihah).
16
4.1.2 FLOWER STAGES
Five different time points were set through the flower development, starting from the bud and
finishing at the fully developed flower. The criteria for each stage were based on the previous
evaluation of the colour formation of two Oriental cultivars: 'Snow Queen' (white) and 'Robina' (pink)
made by Nur Fatihah. The overview of the stage criteria can be found in the Table 3.
Table 3. Overview of the flower
stages
Stage
Bud length [cm]
1
2–5
2
5.1 – 8
3
8.1 – 10
4
>10
5
open flower
4.1.3 GENES
Genes selected for gene expression analysis are involved in the anthocyanin biosynthesis pathway,
with the focus on a branch leading to the cyanidin 3-glucoside (pink to red pigmentation). Seven
structural genes (CHSa, CHSb, CHIa, CHIb, F3H, DFR and ANS) and three transcription factor genes
(bHLH2, MYB12 and MYB15) involved in the red anthocyanin biosynthesis pathway were chosen for
the analysis. Genes encoding transferases were not included as the coloured products are obtained
before the transferases are needed in the pathway.
4.2
METHODS
4.2.1 FLOWER BUDS SAMPLING
Although differences in gene expression between the inner and outer tepals of flowers were reported
(Park et al. 2003) in this experiment the inner and outer tepals are combined. The reasoning behind
this decision is that it is not clear in the early stages which tepals are from the inner and which from
the outer layer and it is even more difficult to separate them once the material is frozen. This is why it
was decided that the consistency in sampling between different stages is of a higher importance.
4.2.2 GRINDING
Part of the buds were ground for further RNA and genomic DNA (gDNA) isolation. Grinding was done
in mortars with liquid nitrogen to ensure that the genetic material will stay intact. The ground tissues
have been stored in a -80°C freezer.
4.2.3 gDNA ISOLATION
One sample from each cultivar was used for gDNA isolation with DNeasy® Plant Mini Kit (250) Cat. No.
69106 according to the manufacture’s instruction. Isolated gDNA was further used as a template in
17
PCR in order to – next to cDNA analysis - confirm the /presence/absence of the genes of interest in the
cultivars.
4.2.4 RNA ISOLATION
First the evaluation of a CTAB protocol (Appendix I), CTAB protocol with RNA cleanup (Cat. No. 74204,
according to the manufactures instruction) and RNeasy mini kit (cat. No. 74106; according to the
manufactures instruction with minor changes: in step 5 – 7 the centrifuge time was extended to one
minute and in step 9, after adding 30µl of RNase-free water, the sample is left standing for one minute
at room temperature and for some of the samples the last step was repeated with a use of the elute
instead of RNase-free water) was performed. Gel electrophoresis and NanoDrop™ analysis were used
for RNA quantity and quality verification. The best method (RNeasy mini kit) was used for RNA
isolation from all the samples.
4.2.5 DNase TREATMENT AND cDNA SYNTHESIS
The RNA concentration was adjusted to 1000ng, 500ng or 250ng (depending on the availability of the
sample). Then DNase treatment (reagents provided by Invitrogen Thermo Fisher Scientific) and cDNA
synthesis (Bio-Rad iScript cDNA synthesis kit) were performed according to the provided protocol
(Appendix II). In the final step after cDNA synthesis, 1000ng samples were diluted up to 200µl, 500ng
samples up to 100µl and 250ng samples up to 50µl in order to obtain the same final cDNA
concentration.
4.2.6 PRIMERS DESIGN
Primers were designed in Primer3Plus (http://primer3plus.com/) based on the lily sequences found in
the Genbank database (http://www.ncbi.nlm.nih.gov/genbank/). When several sequences were
available an alignment was made and primers were designed for the most conserved regions close to
the 3’-end of cDNA sequence. List with all the primers from this study can be found in the Appendix III.
Primers specificity was confirmed by sequencing the amplified products from cultivars and aligning the
sequences to the genes of interest.
4.2.7
GENE EXPRESSION ANALYSIS BY RT-qPCR
Three replicates of each cultivar and time point combination were sampled and used as biological
replicates for gene expression analysis with additional two technical replicates per each biological
replicate. Some of the replicates were used as a source material for gene sequence determination.
The expression of seven genes (CHSa, CHSb, CHIa, CHIb, F3H, DFR and ANS) and three transcription
factors (bHLH2, MYB15 and MYB12) with GAPDH as a reference gene was determined for every
sample in a Bio-Rad C1000 Touch™ qPCR machine. The protocol can be found in the Appendix IV. For
the determination of the relative gene expression (RGE) the 2 –δCt calculation method was used (see
below) and primer efficiency was assumed to be 1.
; where Cq is a number of cycles from RT-qPCR machine
18
4.2.8 STATISTICAL ANALYSIS OF RELATIVE GENE EXPRESSION
All statistical analyses were performed separately for each of the genes. First the raw data from qPCR
output was transformed with a formula = –(Cqgene-CqGAPDH). Two-way ANOVA and significance analysis
with a use of l.s.d. values were performed on these values as the residues had equal distribution and
normality assumption was met. The means for each cultivar x stage combination value (–(CqgeneCqGAPDH) were all transformed by the formula 2x, where x was the mean. This way the relative gene
expression was calculated. The output of ANOVA was used despite blank values (where the expression
was not detected), because it was assumed that the analysis gives a good estimate of l.s.d. value for all
treatments which showed any expression level (non-zero values for gene expression). Nevertheless it
is not an accurate method to analyse the differences with the treatments having no expression (zero
values).
This is why in order to accurately analyse the factor combinations in which the expression was equal
to zero, a separate one-tailed t tests were performed comparing each non-zero gene dataset to zero.
For t value calculation standard deviation of the whole gene data set was used (due to much higher
number of degrees of freedom and so higher confidence). For that reason t value was the same for
each of the treatment combinations within one gene. It means that if t value is higher than t critical for
each data set, then the data set contains enough treatments which are statistically significantly
different from zero. If the value is lower than the critical t, it does not exclude the possibility of having
some values significantly different from zero, but overall most of them are not.
Correlation analysis between RGE of transcription factors and the structural genes were made in R
with a use of Spearman correlation method on the same data set as used for the statistical tests.
4.2.9 SEQUENCING
Amplified products of qPCR primers and long-product primers were sent for sequencing to GATC
Biotech company. To ensure the purity of samples sent for sequencing (both short and long
fragments) several different purifying methods were used depending on the needs of individual
samples. The ones with primer dimers were cleaned with Microspin™ G-50 Columns (protocol can be
found in Appendix V). Samples with unspecific bands were purified by cutting the gel and gel
extraction (Min Elute® Gel Extraction Kit Cat. Nr.28606 according to the manufacture’s protocol).
Samples from qPCR plate were cleaned with QIAquick PCR purification kit (Cat. No. 28106) according
to the manufacture’s protocol.
4.2.10 SEQUENCE ALIGNMENT AND STRUCTURE PREDICTIONS
Quality of the nucleotide sequences was manually checked in Chromas. Then the sequences were
aligned in SeqMan Pro and translated to predicted protein sequences with a use of a translator
(http://www.fr33.net/translator.php). These sequences were then used as a base for 3D secondary
structure prediction in Phyre2 (http://www.sbg.bio.ic.ac.uk/~phyre2) and for 3D structure alignments
PDBViewer was used.
19
5
5.1
RESULTS
EVALUATION OF RNA ISOLATION PROTOCOL
Three different isolation methods (CTAB protocol, CTAB with RNA cleanup and Qiagen RNeasy mini
kit) were tested, with varying results (Table 4; Figure 3A,B and C respectively). CTAB protocol overall
resulted in the highest concentrations, but the lowest quality (A260/280 and A260/230 ratio have to
be above 1.8). The other two methods seem to have comparable results of lower concentration but
high quality. They both are costly, however RNeasy mini kit is much more time-efficient than CTAB +
RNA clean up. This is why RNease mini kit was chosen for the RNA isolation in this study.
Nevertheless, DNA contamination was still present (see Figure 3B and C). Therefore, DNase
treatment was applied. After the DNase treatment DNA was no longer detectable on the gel (Figure
4).
Table 4. NanoDrop™ analysis for different RNA isolation methods
Sample number
Concentration
A260/280
A260/230
2
213.7
1.63
1.41
4
166.8
1.71
1.32
5
277.0
1.68
1.54
6
523.9
1.67
1.43
10
212.6
1.77
1.39
15
224.1
1.66
1.33
26
100.3
2.09
1.83
8
192.7
2.10
2.33
16
271.1
2.10
2.30
18
52.8
2.07
2.01
22
91.4
2.08
1.48
24
250.0
2.08
2.16
1
74.1
2.03
1.96
7
96.1
2.05
2.07
9
102.1
2.06
1.83
17
242.6
2.04
2.24
20
120.3
2.00
2.19
CTAB protocol
CTAB + RNA clean up
Rnease mini kit
23
152.5
2.04
1.48
Sample numbers were assigned based on the order of grinding
the frozen material. The whole set includes more samples from
other cultivars than analysed.
20
Figure 3. A RNA samples isolated with CTAB protocol B RNA samples isolated with CTAB protocol and purified on
the Microspin™ G-50 Columns C RNA samples isolated with RNeasy mini kit. NC – negative control
Figure 4. Representative RNA samples after DNase treatment with 1kb ladder.
5.2
GENES PRESENCE
A series of PCRs was performed on cDNA and gDNA with the primers specific for the genes of interest.
Amplified fragments – together with the data obtained from qPCR analysis – suggest that all analysed
Oriental lilies (‘Rialto’, ‘Perth’ and ‘Gran Tourismo’) carry all the structural genes as well as all three
transcription factors. ‘Lincoln’ seems to be missing DFR and F3’H gene as well as bHLH2 transcription
factor, since no amplicon of these genes could be obtained (Table 5). Different reaction conditions did
not change the outcome.
21
Table 5. Overview of the gene presence and expression in four cultivars. The expression level of each gene is an
simple average over all stages and replicates per cultivar.
Expression level
Gene
Gene
Cultivar
presence
1
2
3
4
CHSa
‘Lincoln’
+
+
‘Rialto’
+
+
‘Perth’
+
+
‘Gran Tourismo’
+
+
CHSb
‘Lincoln’
+
+
‘Rialto’
+
+
‘Perth’
+
+
‘Gran Tourismo’
+
+
CHIa
‘Lincoln’
+
+
‘Rialto’
+
+
‘Perth’
+
+
‘Gran Tourismo’
+
+
CHIb
‘Lincoln’
+
+
‘Rialto’
+
+
‘Perth’
+
+
‘Gran Tourismo’
+
+
F3H
‘Lincoln’
+
+
‘Rialto’
+
+
‘Perth’
+
+
‘Gran Tourismo’
+
+
F3’H
‘Lincoln’
‘Rialto’
+
‘Perth’
+
‘Gran Tourismo’
+
DFR
‘Lincoln’
‘Rialto’
+
+
‘Perth’
+
+
‘Gran Tourismo’
+
+
ANS
‘Lincoln’
+
+
‘Rialto’
+
+
‘Perth’
+
+
‘Gran Tourismo’
+
+
bHLH2
‘Lincoln’
‘Rialto’
+
+
‘Perth’
+
+
‘Gran Tourismo’
+
+
MYB15
‘Lincoln’
+
+
‘Rialto’
+
+
‘Perth’
+
+
‘Gran Tourismo’
+
+
MYB12
‘Lincoln’
+
+
‘Rialto’
+
+
‘Perth’
+
+
‘Gran Tourismo’
+
+
Expression levels: 1 - Hardly expressed (0 > x < 0.1 fold increase); 2 – Lowly expressed (0.1 < x > 0.5 fold increase); 3 Expressed (0,5 < x > 2 fold increase); 4 - Highly expressed (>2 fold increase).
Plus indicates the presence of a gene and/or its expression. Minus indicates absence of the gene in the genome.
22
5.3
SEQUENCE ANALYSIS
Sequence fragments were obtained from all the analysed genes. Their variation in length depended on
the primers used and the quality of the sequence reads. Data obtained was used to confirm gene
presence and to increase the amount of data available from Oriental and Longiflorum lilies.
5.3.1 CHSa
As the quality of gDNA reads was much lower than of cDNA, only the latter was taken into account. In
the qPCR fragment consisting of 230bp there are several single nucleotide polymorphisms (SNPs)
among the cultivars, two of them causing an amino acid (AA) change in position 918+919 and position
1058 (Figure 13). It seems that ‘Lincoln’ is fairly similar to the Oriental lilies, however – analogously to
MYB15 – it is difficult to draw any strong conclusions based only on such a short fragment.
Figure 13. Polymorphisms of CHSa. Red rectangles indicate two AA changes.
5.3.2 CHSb
First thing to notice is that in the gDNA read of ‘Lincoln’ a 78bp sequence is present, which looks very
similar to the one found by Suzuki et al. (2015) in some of the L. speciosum plants (Figure 14). The
most likely explanation is that sequence is an unspliced intron. However ‘Lincoln’ appears to be
missing four nucleotides from its intron sequence.
Figure 14. An intron sequence of CHSb. Red rectangle indicates the intron sequence present in ‘Lincoln’ and two
L. speciosum cultivars. Black rectangles indicate the region with missing nucleotides in ‘Lincoln’.
The AA sequence of two Oriental lilies (‘Gran Tourismo’ and ‘Rialto’) is exactly the same (Appendix VI).
AA sequence of Perth was not determined, as no long nucleotide sequence was obtained. 'Gran
Tourismo' and 'Rialto' differ most from Montreaux (18 AA change), much less from ‘Sorbonne’ (three
AA change) and L. speciosum (single differences from allele nr. 2 of each phenotype). ‘Lincoln’ is also
different from Montreaux (17AA change), but it is less similar to ‘‘Sorbonne’’ (7 AA change), and all
23
three phenotypes of L. speciosum (7, 8 and 6 AA change from red-red, red-yellow and white-yellow
alleles respectively plus single differences from alleles nr. 2 of each phenotype).
5.3.3 CHIa
Aproximately 75% of the CHIa gene length in each of the four cultivars was sequenced in this
experiment. It seems that each of the four cultivars has two alleles of CHIa (Appendix VII). ‘Perth’ and
‘Rialto’ do not seem to overlap, which would indicate that at least three, but possibly four different
alleles are present in the Oriental hybrid group. It is difficult to draw a conclusion about ‘Gran
Tourismo’, as only a small-sized qPCR fragment was sequenced. Nevertheless it seems that ‘Gran
Tourismo’ can have at least one additional allele, different from the two other cultivars. Although it is
not surprising that ‘Lincoln’ has two alleles which are much different from the representatives of the
Oriental lilies, it was not expected to find two SNP sites with three different nucleotides possibilities
(position 216 [A/G/T] and 255 [G/A/T]). Only one or two nucleotides at once occur in each read (Figure
8). In total 13 polymorphic sites were found in all four cultivars, three causing synonymous and 10
non-synonymous AA changes (Appendix VII).
Figure 8. Alignment of CHIa sequences. Two regions in which three different nucleotide reads were found in
Lincoln. Red rectangles indicate sites with polymorphic nucleotides. The alignment with all polymorphic sites can
be found in Appendix VII.
24
Additionally to non-synonymous AA substitutions, it seems that ‘Perth’, ‘Rialto’ and ‘Lincoln’ (this part
of the sequence was not determined for ‘Gran Tourismo’) have a lysine duplication at the end of the
sequence when compared with L. speciosum (Figure 9). Although the sequence quality of ‘Rialto’ can
be questioned, the other two cultivars have very clear reads.
Figure 9. CHIa output of the sequence alignment and sequence reads. The red rectangles indicate the region in
which AA duplication occurred.
Comparison of the protein sequence between our cultivars and L. speciosum (Appendix VI) reveals
that ‘Lincoln’ differs from all the other sequences by five unique AA changes and one change common
with ‘Perth’. ‘Perth’ has one and ‘Rialto’ three additional unique AA changes. Sequence of ‘Gran
Tourismo’ is the only sequence from our cultivars which is 100% identical with red-red and whiteyellow phenotype of L. speciosum. None of the AA changes seem to influence the predicted 3D
structure of the proteins, which suggest CHIa is functional in all cultivars (Figure 10). The only
difference in 3D structure is a missing structure in ‘Gran Tourismo’ in top left corner, which
corresponds to the undetermined part of its nucleotide sequence end. Structures of ‘Perth’, ‘Rialto’
and ‘Lincoln’ were compared with the predicted protein structure of red-red1 phenotype to evaluate a
possible change due to a missing AA. No structural differences were found. Two highest hits from
domain analysis are chalcone isomerase (55%, 52%, 53% and 55% identity for ‘Lincoln’, ‘Rialto’, ‘Perth’
and ‘Gran Tourismo’ respectively) and chalcone-flavonone isomerase 1 (61%, 59%, 60% and 62%
identity for ‘Lincoln’, ‘Rialto’, ‘Perth’ and ‘Gran Tourismo’ respectively). The relatively low identity is
probably cause by a domain template originating from A. thaliana CHI gene, as the sequence
comparison of the chalcone isomerase domain originating from L. speciosum (A0A0P0UVN7 in Uniprot
database) showed much higher sequence similarity (data not shown).
25
Figure 10. Predicted 3D protein structure of CHIa for all four cultivars. The colouration indicates the N to C
direction of the protein starting with red colour at N terminus and reaching blue at the C terminus. GT stands for
Gran Tourismo.
Pocket detection in a 3D model revealed that most of the AA changes does not influence the detected
pocket (in which most probably the active site is located). There is only one AA polymorphism in
position 183 which is a part of the pocket and divides four cultivars into two groups, ‘Lincoln’ with
‘Perth’ and ‘Gran Tourismo’ with ‘Rialto’ (V to I change). The other SNP is not a part of a pocket (Figure
11).
26
Figure 11. 3D alignment between CHIa from all four cultivars. Blue area indicates the identical alignment,
colourful AAs are polymorphic. Long green sequence is an N terminal end which was determined for all but ‘Gran
Tourismo’. White AAs belong to the predicted pocket. Two blue AAs encircled in red are polymorphic (positions
129 and 184 of this alignment)and ant the left on is a part of the pocket (position 184). Residues are hidden to
keep the clarity of an alignment.
5.3.4 CHIb
From CHIb only qPCR sequence data were obtained. In a 223bp long fragment ‘Lincoln’ has six SNPs,
one common with ‘Rialto’, one with L. speciosum and four unique ones. All SNPs except one unique
one are synonymous (SNP in a position 481 causes an AA change from methionine to isoleucine)
(Figure 12). Lincoln is the only cultivar from this alignment which carries isoleucine instead of
methionine.
27
Figure 12. . CHIb alignment between different phenotypes of L. speciosum and cultivars from this study. Red
rectangle indicates the non-synonymous SNP.
5.3.5 F3H
Long fragments were obtained from all three Oriental lilies. Several polymorphic nucleotides were
found within cultivar ‘Rialto’ (Figure 15). First three (position 233 [A/C], 275 [G/A] and 395 [A/G]) are
also shared with ‘Gran Tourismo’. The subsequent four (566 [T/C], 572 [C/G], 710 [T/C] and 761 [T/C])
are in the sequence region which was not determined for ‘Gran Tourismo’, so the presence of
polymorphisms within that cultivar cannot be confirmed or denied. All substitutions except one
(233bp) do not cause AA changes.
Despite the few nucleotide differences within these cultivars the protein sequence of ‘Rialto’ is
comparable with ‘Perth’, ‘Gran Tourismo’ as well as L. speciosum. Two other Oriental lilies (‘Gran
Tourismo’ and ‘Perth’) differ from L. speciosum by one AA only.
According to Uniprot database the key domain in F3H protein is Fe2OG dioxygenase. According to
the 3D modelling the predicted proteins structures of F3H from our cultivars have 22 – 23% identity
with this domain. However the three protein sequences of Fe2OG dioxygenase domain found in the
Uniprot database originating from Lilium hybrid division I and two from L. speciosum were aligned to
the F3H gene sequence from the analysed cultivars and they showed almost 100% identity (one AA
difference) (Appendix VI). At the first sight this result looked contradictory, but after a closer
investigation it was found that the sequence domain used by Phyre2 originates from Arabidopsis
thaliana, not Lilium. This explains poor identity as the domain sequence from Arabidopsis thaliana
differs from the one from Lilium. In 3D structure also a transmembrane helix was predicted for the
F3H gene in all three cultivars at the C-terminal end of the determined protein sequence (at position
249-264), which is also close to the C-terminal end of the gene.
28
Figure 15. Several SNPs present in F3H in ‘Rialto’ and ‘Gran Tourismo’. Red rectangles indicate the
polymorphic sites. Yellow background marks the read area with lower peak intensities.
Figure 16. Predicted 3D protein structure of F3H gene. The colouration indicates the N to C direction of the
protein starting with red colour at N terminus and reaching blue at the C terminus. Red helix is not present in
'Gran Tourismo', because this fragment of the sequence was not determined. GT stands for 'Gran Tourismo'.
5.3.6 F3’H
Short PCR fragments were obtained and sequenced from all four cultivars (with use of F3’H4 primer
pair, see Appendix III), however the same primer pair surprisingly failed to amplify in qPCR reaction.
Sequences from ‘Gran Tourismo’, ‘Perth’ and ‘Rialto’ successfully aligned with high sequence similarity
(90 – 99%) to the reference F3’H gene (AB201532.1). The sequence from ‘Lincoln’ showed only 65%
similarity with the corresponding fragments in Oriental lilies, which suggested nonspecific
amplification. This result was confirmed by resequencing. BLAST search did not result in any significant
similarities. Those results suggest that F3’H is present in all three Oriental lilies, but not in ‘Lincoln’.
5.3.7 DFR
Several SNPs were found in all three Oriental lily cultivars, some of them causing non-synonymous AA
changes. In protein sequence alignment three AA changes were found in which all Oriental lilies (‘Gran
Tourismo’, ‘Perth’ and ‘Rialto’) have the AA profile in common with red-red phenotype of L.
speciosum. Two other AA polymorphisms were identical for Oriental lilies and the white-red
phenotype. Nevertheless, most of the AA polymorphisms (11) formed two groups within which the
SNPs were identical, one with ‘Gran Tourismo’, ‘Perth’ and red-red phenotype and the other with
29
‘Rialto’ and white-red cultivar. Those sequence similarities stand in line with the phenotype of Oriental
lilies. Additionally – within Oriental lilies – three unique AA polymorphisms of ‘Rialto’ and one of ‘Gran
Tourismo’ were found (Appendix VI).
Prediction of the 3D structure (Figure 17) showed several structural differences between ‘Rialto’ and
two coloured cultivars. One difference between ‘Gran Tourismo’ and ‘Perth’ was also found. The
green helix in the bottom right corner is longer in ‘Gran Tourismo’ than in ‘Perth’, however the
sequence in that region is exactly the same in both cultivars. It is possible that this region is more
structure-flexible and it can fold in two different ways, or it can also be a mistake of the program.
Main differences between ‘Rialto’ and the other two cultivars are: in ‘Rialto’ there is a connection
between yellow and orange zone, while there is none in ‘Perth’ and ‘Gran Tourismo’; In the yellow
part ‘Rialto’ has two sheet pieces between two helixes, while only one sheet is formed in the other
two; green and bright blue helix both have an additional turn within itself in ‘Rialto’; in green part two
helixes (the one with the turn and the subsequent one) is connected only by a sheet, while the other
cultivars have an unstructured part. Overall the DFR protein of ‘Rialto’ seem to be more compact than
the other two.
The two highest hits from the domain analysis are dihydroflavonol 4-reductase (65% similarity in all
three cultivars) and anthocyanidin reductase (42% similarity for all three cultivars). The protein pocket
was detected in all three cultivars. The active site and substrate binding site was only detected in
‘Perth’ and ‘Gran Tourismo’. Nevertheless, the AAs of these two sites are not mutated in ‘Rialto’. In L.
speciosum, in both red-red alleles and in one white-red allele active site and substrate binding site
were detected. Additionally in L. speciosum a NADP binding site was detected, starting from a part of
the sequence which was not determined for Oriental lilies.
Figure 17. Prediction of 3D protein structure of DFR in Oriental lilies The colouration indicates the N to C
direction of the protein starting with red colour at N terminus and reaching blue at the C terminus. GT stands for
'Gran Tourismo'.
5.3.8 ANS
Due to a discrepancy in cultivar ‘Rialto’ between the long and qPCR fragment reads the quality of both
was checked. The quality of the reverse read of the long fragment was better than of the qPCR
fragment, and this is why the first one was assumed to be the correct one (Figure 18).
30
Figure 18. Comparison of the ANS gene read quality in cultivar ‘Rialto’.
After an alignment a missing nucleotide was found in the ‘Gran Tourismo’ sequence. However, after a
closer investigation it seems to be only a sequencing error (Figure 19).
Figure 19. Comparison of an alignment with sequence chromatogram of ANS from ‘Gran Tourismo’.
After the sequence correction an alignment of all sequences obtained in this study was made.
Without taking into account a few mismatches at the very end of the sequences, within ‘Gran
Tourismo’, ‘Perth’ and ‘Rialto’ only two SNPs were found. Unfortunately a long fragment of ANS
sequence couldn’t be obtained from ‘Lincoln’. In a 126bp long qPCR fragment four SNPs (all change
into A) were found (Figure 20). This suggests a high density of SNPs between ‘Lincoln’ and Oriental
lilies. Sequences from Lilium ceruum (L. ceruum) and white-red allele from L. speciosum showed over
90% similarity with each other but only 70% similarity with the rest of the sequences (including the
Oriental lilies).
31
Figure 20. Fragment of an alignment of the ANS gene in Oriental lilies and L. speciosum which shows the SNPs in
'Lincoln'.
After translation of a long fragment to a protein sequence all three Oriental lilies have identical
sequences with white-yellow, white-red and one allele of red-red phenotype of L. speciosum. One AA
difference was found between Oriental lilies and the other allele of red-red phenotype. The Oriental
lilies tested by us are less similar to Montreaux, L. ceruum and ‘Sorbonne’ (six, five and three AA
differences respectively). The reason why the protein sequences of L. ceruum and white-red allele
align well with the rest of the sequences is that there is an untranslated fragment, which is probably
an intron and it was removed from the protein sequence (Appendix VI).
5.3.9 bHLH2
Next to qPCR fragments, only one long fragment of bHLH2 was sequenced, originating from ‘Gran
Tourismo’. No amplicon from ‘Lincoln’ could be found as no primers attached to it. An alignment with
several L. speciosum sequences and one Asiatic hybrid cultivar ‘Montreux’ was made. qPCR sequences
of all three cultivars are more similar to L. speciosum. ‘Perth’ and ‘Rialto’ showed 100% similarity,
while qPCR fragment of ‘Gran Tourismo’ differs from L. speciosum by two nucleotides. The long
fragment from ‘Gran Tourismo’ has higher concentration of SNPs than the short fragment (29 SNPs
per 542bp while in qPCR fragment two SNPs per 231bp). Additionally ‘Gran Tourismo’ has a threenucleotide deletion at 1270bp, which results in one missing asparagine (Figure 5).
Figure 5. An assembly between bHLH2 of six L. speciosum sequences, one ‘Montreux’, long fragment from ‘Gran
Tourismo’ and separate forward and reverse reads of ‘Gran Tourismo’ from which an alignment was made. Red
rectangle indicates an area with missing codon. Although the reverse read has a lower quality in that region it is
still consistent with high-quality forward read.
32
SNPs found in ‘Gran Tourismo’ influence its protein sequence by causing eight amino acid (AA)
substitutions (Appendix VI). There are 11 AA differences, where ‘Gran Tourismo’ is identical with
either L. speciosum or ‘Montreux’ cultivar. At the end of the long protein fragment at the position 403
there is an asparagine deletion, which corresponds to a missing codon in position 1270 (Figure 5).
5.3.10 MYB12
First thing to notice after making an alignment is that sequences from some representatives of division
VII have a somewhat similar insertion, absent in other cultivars (Figure 6). It is possible that this
sequence is a residue of an intron. No residual sequences were found in the analysed Oriental
cultivars (which also belong to division VII).
Figure 6. MYB12 alignment of several different cultivars. In hybrid division VII an insertion is present.
In order to keep clarity of the alignment the hybrids from division VII were removed. Short qPCR
fragments from all four cultivars were generally highly similar to the other sequences. In position 538
of this alignment ‘Gran Tourismo’ had a polymorphism identical with red-red1 phenotype, which
causes a substitution of histidine with asparagine (Figure 7). In position 570 ‘Perth’ and ‘Rialto’ have
the same polymorphism as red-red2 and white-red 1 phenotype, while ‘Gran Tourismo’ and ‘Lincoln’
has the polymorphism of the other cultivars (synonymous mutation). Additionally ‘Gran Tourismo’ has
one unique SNP in position 600 causing an AA change from glutamine to histidine and ‘Lincoln’ in
position 622 causing a substitution of cysteine with glycine (Figure 7). An additional alignment was
made with L. auratum, in which approximately half of the SNPs present in long fragment of Gran
Tourismo were identical with L. auratum sequence The other half was either identical with L.
speciosum or unique(data not shown).
Figure 7. Polymorphic area of the alignment of MYB12 with sequences from L. speciosum and cultivar
‘‘Sorbonne’’.
33
5.3.11 MYB15
Only qPCR fragments from ‘Gran Tourismo’, ‘Rialto’ and ‘Lincoln’ were obtained. When aligned few
SNPs were found, nevertheless no strong conclusions could be made based on such a short fragment.
Nevertheless, these qPCR fragments proved the presence of MYB15 in each of the three cultivars
(presence in ‘Perth’ was proven by qRT-PCR analysis).
5.4
GENE EXPRESSION
5.4.1 REFERENCE GENE EVALUATION
Three candidate reference genes were analysed (Actin, UBQ4 and GAPDH). All three genes showed a
relatively wide range of Cq values (UBQ4 22,84 – 30.76; Actin 22,37 – 37.33; GAPDH 19.32 – 24.80). As
a concern arose that the initial amount of cDNA differed between the samples due to a DNase
treatment, a trail with samples from a higher extreme (all originating from ‘Perth’) was made. A
change in a procedure for cDNA synthesis was applied. First, the sample was subjected to a DNase
treatment and afterwards the concentration of RNA was determined and based on that result the
amount of RNA for cDNA synthesis was determined. Nevertheless, the results were mostly overlapping
with the previous values (Figure 21). Based on that the assumption was made, that the differences in
the expression of the reference genes is due to other factors than the initial amount of RNA.
Figure 21. Comparison of 1st and 2nd trial cDNA from three top extreme samples (all originating from ‘Perth’).
Numbers indicate flowering stages of the sample, letters a and b indicate technical replicates.
To ensure the most precise analysis of the gene expression at first an average of UBQ4 and GAPDH
was considered to be used. However, for some of the samples over 30 cycles was needed to reach a
threshold for UBQ4 while GAPDH values were placed closer to 20. 20 is a preferable value for a
reference gene as it is in the middle of the machine measuring range. Based on those findings, GAPDH,
despite the wide range of Cq values, was concluded to have the highest expression stability and was
chosen as the reference gene for the gene expression analysis in this study (Figure 22). However its
variation has to be kept in mind when conclusions from gene expression analysis are drawn. For
example an expression of GAPDH in ‘Gran Tourismo’ decreases over time (increasing Cq values) which
34
can cause an overestimation of gene expression level. In ‘Rialto’ stage 5 and ‘Lincoln’ stage 1 the
expression of GAPDH is high (low Cq value) and it can lead to underestimation of the gene expression
level. Nevertheless, neither a pattern nor significant differences in GAPDH expression were observed.
Figure 22. Expression level of GAPDH in all cultivars and stages. GT stands for 'Gran Tourismo'.
5.4.2 PRIMER DIMERS
For few of the analysed genes (GAPDH, CHIb, F3H, ANS and MYB15) primer dimers were detected in
non-template controls (NTC) and in some of the samples in which there was no detectable gene
expression. However in the samples in which expression was detected no primer dimers were formed.
Based on that, it was assumed that the obtained Cq values from expression data are due to the gene
of interest only. Additionally it was assumed that if primer dimers were formed there was no cDNA
template for primers to attach to and the gene expression level was assumed to be zero.
5.4.3 RELATIVE GENE EXPRESSION
The expression of all the genes described in this section is depicted in Figure 23. Significant differences
for each of the genes are shown in a letter code in Table 6. Table 6A shows a cultivar x stage
interaction effect, while 6B and 6C shows the cultivar and stage effect respectively, for the genes in
which interaction effect was not significant. F3’H gene was discarded from the gene expression
analysis as no suitable primers for RT-qPCR were obtained.
35
5.4.3.1 STRUCTURAL GENES
5.4.3.1.1 CHSa &CHSb
In ‘Gran Tourismo’ and ‘Perth’ CHSa is upregulated in stage 4 and 5 when compared with the first
three stages. In ‘Lincoln’ only stage 5 is upregulated up to a similar level as in ‘Perth’. Expression of
CHSa in ‘Rialto’ is hardly detectable. Expression of CHSb in ‘Gran Tourismo’ has the same pattern as
CHSa, lower in the first three stages and higher in stage 4 and 5. In ‘Perth’ CHSb is upregulated in stage
1,4 and 5. CHSb in ‘Lincoln’ and ‘Rialto’ is hardly expressed.
5.4.3.1.2 CHIa & CHIb
Both CHI genes have the highest expression in ‘Gran Tourismo’ stage 4 and 5 reaching approximately
20 and 6 fold increase for CHIa and CHIb respectively. The expression of CHIa in the other three
cultivars (‘Perth’, ‘Rialto’ and ‘Lincoln’) has the same trend as ‘Gran Tourismo’ but much lower
expression. The expression of CHIb is more levelled in ‘Lincoln’ and ‘Perth’. ‘Rialto’ has a very low
expression of CHIb, with an exception for stage 2, which is significantly higher than the subsequent
steps.
5.4.3.1.3 F3H
The expression of F3H is highest in ‘Gran Tourismo’, with the peak in stage 4. In ‘Rialto’ a similar trend
is observed, but with the highest expression peak in stage 3. In ‘Perth’ the expression seems to
gradually increase over the flowering time. In ‘Lincoln’ is more levelled, but it seems to slowly, but
gradually decrease.
5.4.3.1.4 DFR
Expression of DFR in ‘Gran Tourismo’ and ‘Perth’ increases until stage 4 and decreases in stage 5. In
‘Gran Tourismo’ the expression in stage 3, 4 and 5 is significantly higher from stage 1 and 2. In ‘Rialto’
DFR is hardly expressed while in ‘Lincoln’ the DFR gene was not found.
5.4.3.1.5 ANS
Expression of ANS is low in all four cultivars, it doesn’t exceed 2 fold increase. ‘Gran Tourismo’ displays
the highest expression level, with ‘Perth’ following it. In both cultivars expression of ANS increases up
to stage 4 and stays on the same level in stage 5. In ‘Rialto’ and ‘Lincoln’ ANS is hardly expressed.
5.4.3.2 TRANSCRIPTION FACTORS
Correlation plots for the transcription factor bHLH2, MYB15 and MYB12 with the structural genes can
be found in Figure 24, 25 and 26 respectively. The rho values for each cultivar of all of the
transcription factor and structural gene combinations can be found in Table 7.
5.4.3.2.1 BHLH2
Expression of bHLH2 in ‘Lincoln’ was below a detectable level. In all the other cultivars the level of
expression was very low (not exceeding 0.015 folds increase in compare to a reference gene). There is
a trend visible in ‘Rialto’ – and in lower extent in ‘Perth’ – to decrease the expression over time.
Nevertheless the values are very small and differences were not significant (P >0.05). A strong
correlation between bHLH2 and structural genes of Gran Tourismo was found (all rho above 0.75)
(Figure 24, Table 7). bHLH2 was also strongly correlated with CHIb in Rialto (rho=0.80). The rest of the
structural genes from Rialto and all the genes from Perth did not show any strong correlation with
bHLH2.
36
5.4.3.2.2 MYB15
In Oriental lilies the highest peak of MYB15 expression is in stage 4, while in ‘Lincoln’ this peak occurs
in the stage 2. Overall ‘Gran Tourismo’ has the highest expression among these cultivars, ‘Lincoln’ and
‘Perth’ a medium and ‘Rialto’ the lowest. MYB15 expression is highly correlated with all the structural
genes from 'Gran Tourismo', CHIa, CHSa, F3H and DFR from Perth, CHIa, F3H and DFR from Rialto and
F3H from Lincoln. MYB15 is also negatively correlated with CHSa from Rialto and Lincoln (rho=-0.46
and -0.31 respectively) (Figure 25,Table 7).
5.4.3.2.3 MYB12
In all cultivars a trend to have the highest MYB12 expression in stage four was observed (however
MYB12 in ‘Lincoln’ was hardly expressed at any examined stage). In ‘Gran Tourismo’ and ‘Perth’ stage
4 was significantly different from stage 1 and 2. In ‘Rialto’ the difference was observed only between
stage 4 and 1. Only in ‘Gran Tourismo’ a significant difference between stage 5 and 2 was observed, in
other cultivars a decrease in stage 5 returned the level of expression to the initial state from stage 1
and 2. MYB12 is very strongly correlated with the structural genes of Gran Tourismo (rho>0.90 for
each of the structural genes)(Figure 26, Table 7). In Perth the correlation is not strong (rho varies
between 0.346 and 0.699). In Rialto a strong correlation with CHIa (rho=0.82) and DFR (rho=0.80)
occurs, but also a negative correlation with CHIb, CHSa and CHSb (-0.41, -0.59 and -0.45 respectively).
No strong correlation in Lincoln was found.
37
38
39
40
Figure 23. RGE in all cultivar x stage combinations for CHSa (a, b),CHSb (c, d), CHIa (e, f), CHIb (g, h), F3H (i, j), DFR
(k, l), ANS (m, n), bHLH2 (o, p), MYB15 (q, r) and MYB12 (s, t). On the left big scale graphs are presented (a, c, e,
g, i, k, m, o, q and s). On the right side the zoomed graph are presented (b, d, f, h, j, l, n, p, r and t) which were
made for better view on the cultivars with lower RGE. Also please note different scales on CHIa, CHSb, bHLH2
zoom and MYB15 zoom. Scales of these graphs were adapted to the expression levels in order to keep them
readable. Significant differences between cultivars are shown in Table 6.
Table 6 A Statistical significance for the genes in which cultivar x stage interaction is significant. Genes are not
comparable between each other. B Statistically significant differences between cultivars for genes in which
cultivar x stage interaction is not significant. C Statistically significant differences between stages for genes in
which cultivar x stage interaction is not significant. GT stands for ‘Gran Tourismo’.
A
Cultivar
Stage
GT
Genes
CHSa
CHSb
CHIa
CHIb
F3H
bHLH2
MYB15
1
d
efg
ghi
ef
abcde
a
fg
GT
2
d
cdef
fg
def
cde
a
efg
GT
3
e
h
j
g
f
-
hi
GT
4
f
i
k
h
g
b
i
GT
5
e
h
k
h
f
b
g
‘Perth’
1
bc
fg
abcd
cd
ab
e
b
‘Perth’
2
bc
abcd
a
def
abc
de
cde
3
c
def
abc
def
bcde
de
g
‘Perth’
4
d
g
cdefg
def
cde
de
ghi
‘Perth’
5
d
g
fg
de
ef
de
efg
‘Rialto’
1
bc
cdef
bcde
bc
ab
de
a
‘Rialto’
2
ab
bcde
ab
def
abcd
de
c
‘Rialto’
3
a
abcd
hij
b
def
de
cd
‘Rialto’
4
-
abcd
ij
-
ab
d
def
‘Rialto’
5
a
abcd
efg
a
abc
c
ab
‘Lincoln’
1
c
abcd
bcdef
ef
bcde
-
gh
‘Lincoln’
2
c
ab
defg
efg
bcde
-
hi
‘Lincoln’
3
bc
abc
cdefg
fg
abc
-
ghi
‘Lincoln’
4
bc
a
gh
def
ab
-
fg
‘Lincoln’
5
d
bcde
bcdefg
ef
a
-
cd
DFR
ANS
MYB12
‘Perth’
C
B
Cultivar
Gene
Stage
Gene
DFR
ANS
MYB12
GT
a
ANS
c
1
-
-
a
‘Perth’
b
b
b
2
a
ANS
a
‘Rialto’
-
-
a
3
b
b
c
‘Lincoln’
-
-
-
4
b
c
d
5
b
bc
b
41
Figure 24. Correlation between bHLH2 and structural genes. Colours indicate different cultivars. Each of the
cultivars has its own trend line. GT stands for 'Gran Tourismo'.
42
Figure 25. Correlation between MYB15 and structural genes. Colours indicate different cultivars. Each of the
cultivars has its own trend line. GT stands for 'Gran Tourismo'.
43
Figure 26. Correlation between MYB12 and structural genes. Colours indicate different cultivars. Each of the
cultivars has its own trend line. GT stands for 'Gran Tourismo'.
44
Table 7. Rho correlation values for each cultivar between transcription factors and the structural genes.
Correlation
bHLH2
MYB15
MYB12
CHIa
CHIb
CHSa
CHSb
F3H
DFR
ANS
GT
0.937
0.862
0.937
0.887
0.912
0.895
0.753
Perth
-0.175
0.193
-0.164
-0.116
-0.257
0.14
0.077
Rialto
-0.025
0.802
0.238
0.343
0.567
0
0.257
Lincoln
-
-
-
-
-
-
-
GT
0.782
0.707
0.861
0.854
0.85
0.836
0.775
Perth
0.666
0.358
0.776
0.284
0.807
0.874
0.643
Rialto
0.693
0.151
-0.456
-0.295
0.651
0.8
-0.086
Lincoln
0.25
0.565
-0.315
-0.198
0.77
-
0.1
GT
0.957
0.943
0.918
0.989
0.921
0.968
0.929
Perth
0.604
0.346
0.725
0.416
0.671
0.699
0.559
Rialto
0.824
-0.406
-0.591
-0.446
0.409
0.8
1
Lincoln
0.5
0.3
0.3
0.3
0.1
-
-
Rho values were determined with a use of Spearman correlation test. Values above 0.9 are underlined and
marked in bold. Values above 0.75 but lower than 0.9 are marked in bold. Negative correlation values are
written in italic. Minus sign “-“ indicates lack of one of the genes (in case of MYB12 versus ANS in 'Lincoln', not
enough data points were found due to very low expression of both genes). In 'Perth' MYB12 versus ANS a low
amount of data points was used which probably led to an overestimation of the result. GT stands for 'Gran
Tourismo'.
45
6 DISCUSSION
6.1
SEQUENCES AND GENE EXPRESSION
6.1.1 STABILITY OF THE REFERENCE GENE
All three candidate reference genes which were analysed (Actin, UBQ4 and GAPDH) did not show high
stability, but varied from 19.32 to 37.33 cycles. It is however important to notice that those three
genes are considered to have the lowest variation among different tissues in Lilium davidii (tepals
were not evaluated) (Li et al. 2015). In the study of Liu et al. (2016) GAPDH showed quite a wide range
of Cq values, ranging from approximately 16 to 23 cycles and yet in combination with other genes is
considered the best reference combination for drought and salt stress gene expression studies in
Lilium regale (Liu et al. 2016). The variation in Cq values of the reference genes has to be kept in mind,
nevertheless GAPDH is still one of the best options available.
6.1.2 CHSa and CHSb
No strong conclusions could be made for a CHSa nucleotide sequence, as the obtained fragment is
only 230bp long. It does seem that this fragment of the gene is fairly conserved among Oriental and
Longiflorum lilies, but longer reads are needed to confirm this conclusion. In CHSb longer fragments
were obtained from two Oriental lilies and ‘Lincoln’, which account for over 90% of the full gene
length. All three sequences were more similar to ‘Sorbonne’ cultivar and L. speciosum than to
‘Montreux’. ‘Lincoln’ has the lowest similarity level among these three cultivars analysed in this study
(Appendix VI).
The CHSa gene expression in ‘Gran Tourismo’, and partially in ‘Perth’ (only in the last two stages) was
generally higher than in ‘Rialto’. These results stands in line with the results of Shi et al. (2014) for
Magnolia sprengeri in which higher expression was associated with a darker petal colour.
In a study of Yamagishi (2011) the expression of CHSa in pink tepals of ‘Sorbonne’ cultivar was close to
zero in stage 2, comparably high in stage 3 and 4 and very low in stage 5. CHSb had a similar pattern
except that expression in stage 3 was lower than stage 4 (Yamagishi 2011). This somewhat stands in
contradiction with the results obtained in this study, where the high expression in stages 3 and 4 and a
(small) decrease in stage 5 for both CHSa and CHSb genes was only observed in ‘Gran Tourismo’. In
‘Perth’ a small increase in expression was observed in stage 1 (CHSb) and in the last two stages (CHSa
and CHSb). In ‘Lincoln’ CHSa showed a small increase in the last stage, while CHSb was hardly
expressed at any stage. Both CHSa and CHSb in ‘Rialto’ were hardly expressed at any stage (yet still
present in cDNA).
In the study of Suzuki et al.(2015) the expression of CHSa in L. speciosum red-red phenotype and
white-yellow was almost 50x and 1000x higher than the expression in ‘Gran Tourismo’ and ‘Lincoln’ in
the equivalent stage 4, respectively. Expression of CHSb in red-red cultivar more or less matches the
expression level of ‘Gran Tourismo’, but white-yellow cultivar is 5x higher expressed than ‘Lincoln’. The
differences within CHSb could be explained by different reference genes used (UBQ4 by Suzuki and
GAPDH in this study), but that is an unlikely explanation for CHSa. It seems that expression levels of
CHSa differ greatly between different lily species. It could indicate that CHSb, as less variable among
46
different cultivars, is more important in colour determination than CHSa. However to confirm this an
experiment with CHSa knock-out mutant should be performed, to see if the coloured flowers are
developed.
6.1.3 CHIa and CHIb
A presence of three different nucleotide reads in two places in ‘Lincoln’ were most probably caused by
a lower reverse read quality, as after a closer investigation this read has quite a lot of “background
peaks”. In general the activity of CHIa is probably not impaired in any way, as the AA changes found
within the sequence of the Oriental lilies from this study (which accounts for ~75% of the full-length
gene) are outside the active site and they do not influence the 3D structure in any visible way (Figure
10). The only AA change found inside the active site (substitution of valine with isoleucine) is unlikely
to have a big influence either (Figure 11). Both AAs are hydrophobic and have similar preferences for
the secondary structure, the main difference is size, isoleucine is slightly bigger than valine.
Although only short qPCR sequence fragments were obtained from CHIb gene, a difference between
‘Lincoln’ and other cultivars is visible. Presence of four SNPs in a 223bp long fragment suggests a high
SNPs concentration in ‘Lincoln’. Nevertheless longer fragments should be sequenced to confirm these
deductions. The expression pattern of CHIb is mostly similar to CHIa, however the level of expression
is at least twice lower. The expression patterns are in line with the results of Suzuki et al. (2015). .
CHIa expression can be partially explained by the findings of Suzuki et al. (2015), where it was
mentioned that CHIa is probably not regulated by MYB12. Despite significantly higher MYB12
expression in ‘Perth’ than ‘Lincoln’ there is no difference in their expression of CHIa. Additionally
MYB12 in ‘Rialto’ was hardly expressed, while CHIa had the second highest expression among four
cultivars. Another argument supporting this conclusion is that the correlation factor between those
two genes did not exceed 0,8, which also indicates, that even if a correlation exist, it is not very strong
(Table 7).
Summarizing, it is difficult to judge which of the CHI genes may play a more important role in
anthocyanin biosynthesis pathway, as both of the genes display similar expression pattern and none of
them seem to have a disrupted amino acid sequence.
6.1.4 F3H
Based on the sequence reads from this study the conclusion was made that ‘Rialto’ and ‘Gran
Tourismo’ are both heterozygous for F3H gene. The alleles seem to be the same in these two cultivars,
but full-length sequence reads are needed to confirm it (the sequence length determined so far covers
approximately 80% of the gene). The presence of the second allele need confirming especially for
'Gran Tourismo' as its sequence read was shorter of approximately 100bp and did not cover all of the
polymorphic sites found. Furthermore it seems that the F3H gene is a transmembrane protein with a
transmembrane domain close to the C-terminal end of a gene (AAs 249-264). Nevertheless it seems
that the sequence and 3D structure of F3H is well conserved as only one AA substitution was found
between Oriental hybrids and L. speciosum (Figure 16).
Yamagishi (2011) found the F3H expression in ‘Sorbonne’ (pink tepals) to be increasing over stages 2 4 (Yamagishi analysed only stages 2 – 5) and then suddenly decreasing in the last fifth stage. Similar
47
pattern was found only in ‘Gran Tourismo’, however the decrease in the last stage was not as steep as
in ‘Sorbonne’. In ‘Rialto’ the decrease started already in stage 4, in ‘Lincoln’ in stage 2 while in ‘Perth’
only steady increase was observed. Those results are surprising, especially for ‘Perth’, as a pattern
similar to ‘Sorbonne’ was expected due to its similarity in colour and affiliation to the same lily
division. In the study of Suzuki et al. (2015) on L. speciosum the F3H expression in red-red and whiteyellow phenotype is on a comparable level, while the expression in white-red phenotype is much
lower. In our study the expression of the phenotypically corresponding cultivars the expression of
Rialto (white-red phenotype) is on a similar level as in L. speciosum, ‘Gran Tourismo’ has indeed
higher expression than ‘Rialto’, but ‘Lincoln’ (white-yellow phenotype) has the expression on the
similar or even lower level than ‘Rialto’ (Figure 23). Nevertheless, assuming that dihydroquercetin
(product of the reaction catalysed by F3H) is present in Lincoln as it is present in another Longiflorum
cultivar ‘Snow Queen’ (data from Nur Fatihah) the conclusion would be that F3H expression level does
not seem to be a limiting factor of the anthocyanin biosynthesis within the analysed cultivars.
6.1.5 DFR
Suzuki et al. (2015) found a premature stop codon in the DFR sequence of L. speciosum and suggested
this mutation occurs in the white flowered lilies with yellow tepals. In this study 59% of the gene
sequence was determined overlapping with the region in which the mutation in L. speciosum is
present. This mutation was not found in the sequence fragments of any of the Oriental lilies in this
study (Figure 15). That implies a stop codon mutation mentioned by Suzuki et al. (2015) is not present,
which is in line with the phenotype of these lilies (all of them have red anthers). The only cultivar in
which this mutation was expected to be found is ‘Lincoln’. However, based on the results of this study
it was concluded that DFR gene is not present in ‘Lincoln’ cultivar.
DFR is only hardly expressed in ‘Rialto’, which is in line with the findings of (Suzuki et al. 2015). The
expression pattern in Perth differs from a ‘Sorbonne’ cultivar, as in ‘Sorbonne’ a steep decrease
between stage 4 and 5 is observed, while in both coloured Oriental lilies the decrease is much smaller.
The similar expression pattern was expected due to the similar phenotype of these two lily cultivars.
Despite hardly any expression in ‘Rialto’ cultivar, DFR is assumed to be at least partially functional.
Firstly because lilies with red anthers are considered to carry a functional DFR gene (Suzuki et al.
2015). Secondly ‘Rialto’ is lacking a mutation which was found in a NADPH binding site of DFR in
gerbera cultivar, where glycine was changed into cysteine which caused lack of DFR enzymatic activity
(Bashandy et al. 2015). Yet it is possible that its function in ‘Rialto’ is (partially) impaired, as the active
sites were not detected with Phyre2 program, despite the presence of the same AAs in the active site
and substrate binding site. It could be caused by the presence of several other AA changes which
influenced the 3D structure so much, that the protein is unable to bind the substrate anymore (Figure
16).
Summarizing, DFR gene may be a limiting factor in the anthocyanin biosynthesis pathway within Rialto
cultivar, because it carries several AA changes and it is hardly expressed at any flower stage. DFR is not
considered to be a limiting factor in 'Perth' as its protein sequence is identical with 'Gran Tourismo'
and the gene itself is expressed in all stages excluding stage 1.
48
6.1.6 ANS
When the expression patterns of ANS and the phenotypes of the cultivars are compared, the results
are in line with findings of Nakamura et al. (2006), where lowering of the ANS expression caused a
paler petal colour. ANS is hardly expressed in both white cultivars, lowly expressed in pink ‘Perth’ and
the highest expression is observed in ‘Gran Tourismo’. Nevertheless it should be noted that an ANS
expression is in general very low, not exceeding 0.6 fold increase when compared with a reference
gene.
6.1.7 bHLH2 AND MYB15
Sequence analysis suggests that the bHLH2 gene has fragments which are more and less conserved
among different lily cultivars and species. An example of this is the qPCR fragments from all three
Oriental lilies which were highly similar with L. speciosum and ‘Montreux’ sequences while a longer
fragment of ‘Gran Tourismo’ showed a higher concentration of SNPs (higher number of SNPs per
length of the fragment in bp). It seems that the long sequence fragment of ‘Gran Tourismo’ is equally
dissimilar from ‘Montreux’ and L. speciosum, having eight and twelve common SNPs respectively.
Additionally in the longer fragment ‘Gran Tourismo’ has nine unique SNPs (Figure 5). Eight of these
SNPs are non-synonymous, however not much can be said about a possible influence on bHLH2 3D
structure, as no well-matching model could be obtained from Phyre2 and the sequence which was
obtained covers only ~23% of the gene length.
Both Nakatsuka et al. (2009) and Suzuki et al. (2015) suggested that bHLH2 is involved in anthocyanin
biosynthesis as its expression is parallel with anthocyanin formation. bHLH2 expression level
and its patterns over the different flowering stages in ‘Perth’ and ‘Rialto’ were similar to ‘Montreux’
and ‘Connecticut King’ (Nakatsuka et al. 2009). Nevertheless, in this study no clear link in the
expression patterns between bHLH2 and structural genes could be found, as the lowest bHLH2
expression was found in ‘Gran tourismo’, which is the darkest coloured cultivar. Although the
correlation analysis showed that bHLH2 is strongly correlated with the structural genes expression in
‘Gran Tourismo’ (Figure 24; Table 7), it should be kept in mind that bHLH2 expression in ‘Gran
Tourismo’ is very low (Figure 23), which makes it questionable if this correlation result can be trusted.
The same correlation analysis did not show any strong relation within Perth. Only within Rialto the
correlation with CHIb gene was above 0.8 (Figure 24; Table 7).I therefore suggest, that bHLH2 is not
necessary for an induction of any of the structural genes , but – for example – expression of another
bHLH gene or MYB15 is more important.
However an observation was made by Suzuki et al. (2015) that MYB15 is only expressed in tissues in
which anthocyanin accumulation occurs. The results obtained in this study partially contradict these
observations, as the expression in ‘Lincoln’ and ‘Perth’ (white and pink cultivar) are comparable, with
‘Gran Tourismo’ being not much higher than them. Those results indicate that expression of MYB15 is
not necessarily correlated with anthocyanin presence. The correlation between MYB15 gene
expression and the structural genes within ‘Gran Tourismo’ is lower than the one of bHLH2,
nevertheless it is still above 0.75. MYB15 is also more correlated with CHSa and CHIa within 'Perth'. In
'Rialto' CHIa is also correlated with MYB15, but it seems that CHSa has a negative correlation with
MYB15. Similar correlation pattern is observed in 'Lincoln', however the rho values are closer to zero
(weaker correlation).
49
6.1.8 MYB12
Suzuki et al. (2015) found the mutation causing a premature stop codon in white-yellow phenotype of
L. speciosum. As expected, ‘Gran Tourismo’ is lacking this mutation and it has a functional MYB12
gene. Unfortunately sequence fragments of this region from ‘Perth’, ‘Rialto’ and ‘Lincoln’ are not
available. MYB12 was the only gene of which the sequence was found from another (beside L.
speciosum) parental line of Oriental hybrids, which is L. auratum. Based on the alignment of the L.
auratum sequence with a long fragment of ‘Gran Tourismo’ (74% of the full length gene) it was
concluded that MYB12 gene in ‘Gran Tourismo’ is more related to L. auratum than to L. speciosum.
Unfortunately, no sequences from other parental lines of Oriental hybrids are available and no
alignments could be made.
Suzuki et al. (2015) suggested that MYB12 is responsible for inducing expression of several structural
genes, such as CHSa, CHSb, CHIb, F3H, F3’H, DFR and ANS. In our hands, the expression patterns of
MYB12 and the above mentioned genes mostly fit this suggestion in ‘Gran Tourismo’ and ‘Perth’.
Nevertheless CHIb in ‘Gran Tourismo’ doesn’t show a drop in expression in stage 5, as it would be
suggested by a sudden drop of MYB12 expression and in ‘Perth’ it is barely increasing over time.
Additionally in ‘Perth’ F3H seems to be outside of the pattern, as its expression only increases over
time, without a drop in stage 5. The same theory does not seem to apply to ‘Lincoln’, where
expression of MYB12 was detected with a peak in stage 4 (although one has to keep in mind the
expression in ‘Lincoln’ is very low), while only CHSa and CHSb have a peak in stage 5. The other genes
do not seem to fit to the pattern. For example F3H steadily decreases over time and CHIb and ANS are
hardly expressed at all. All these deductions stand in line with the correlation analysis, where the
strong correlation between MYB12 and the structural genes was only found within ‘Gran Tourismo’.
Lower, but still positive correlation was observed in Perth, while in Rialto CHSa, CHSb and CHIb
showed a negative correlation and DFR and ANS did not have enough data points for a reliable
analysis. MYB12 expression in Lincoln did not seem to be linked to the expression of the structural
genes.
6.2
DIFFERENCE BETWEEN ‘GRAN TOURISMO’ AND ‘PERTH’
As ‘Gran Tourismo’ is a dark red cultivar and ‘Perth’ a pink one it was expected to see differences in
either gene expression or gene sequence which could explain this difference in phenotype. Overall, all
the genes except bHLH2 follow approximately the same pattern in both coloured cultivars, but the
expression level is always much higher in ‘Gran Tourismo’ (from 2 fold higher in MYB15 up to 100 fold
in CHSb). The difference only in the expression level is a likely explanation for the difference in
phenotype, as high expression of transcription factors will highly induce the expression of the
structural genes needed for anthocyanin biosynthesis. However it raises a question why are the
transcription factors so highly expressed. Several reasons may explain this phenomena, such as
sequence differences within the gene and/or its promotor, or for example presence of other
transcription factors which influence the expression of both MYB15 and MYB12 and supress the
expression of bHLH2 in ‘Gran Tourismo’.
6.3
WHY IS ‘RIALTO’ WHITE
50
The conclusions obtained by Suzuki et al. (2015) with respect to the origin of the white colour of
‘Rialto’ tepals is in line with the results obtained in this study. Although the point mutation from
MYB12 couldn’t be observed due to insufficient sequencing data, the expression of the genes – which
are presumably induced by MYB12 – were found to be very low (below 1 fold increase). It might be
interesting to find out if F3H is only regulated by MYB12 gene, as its expression is relatively high in
comparison to other genes in this cultivar. Also based on the correlation analysis it is highly correlated
with MYB15 in all the cultivars, but its correlation with MYB12 is high only in ‘Gran Tourismo and
‘Perth’. CHIa – which is considered to have a different transcription factor – is the only gene which
expression is higher than 1 fold increase compared to the reference gene.
In relation to late stage genes there is some discrepancy about DFR protein function. As mentioned
before it lacks any of the crucial mutations which would impair its function and it does have red
anthers, which indicates a functional anthocyanin pathway. However, its structure is quite different
from both ‘Gran Tourismo’ and ‘Perth’. Also Phyre2 did neither recognise an active site nor the
substrate binding site. In case the DFR enzymatic activity is impaired it could be another factor
causing ‘Rialto’ to have white flowers.
6.4
WHY IS ‘LINCOLN’ WHITE
Based on the findings of our study the most likely cause of white flower colour in the Longiflorum
cultivar ‘Lincoln’ is the lack of two structural genes, F3’H and DFR. The main argument for assuming a
lack of F3’H gene is that the primer pair used amplified a F3’H gene fragment in all three Oriental lilies,
while in ‘Lincoln’ it only amplified an aspecific fragment with a similar sequence length. The
assumption of the absence of DFR is supported by two arguments. Firstly the primer pairs (for both
qPCR and long fragments), which successfully amplified DFR in other cultivars did not work in ‘Lincoln’,
even when the conditions of the PCR were altered. The second argument behind this conclusion is
that ‘Lincoln’ flowers have yellow anthers, which according to Suzuki et al. (2015) is linked with a
disruption of DFR. Moreover, since no proof of the DFR existence in ‘Lincoln’ could be found, it is
reasonable to assume that the gene itself is missing from the genome.
The third gene which is considered missing from the genome of 'Lincoln' is bHLH2. However, despite a
lack of bHLH2 transcription factor, early stage genes are expressed in ‘Lincoln’, which could be
explained by the expression of MYB15 transcription factor (Suzuki et al. 2015). ‘Lincoln’ also carries
and expresses MYB12 transcription factor, which is known to induce late stage genes (Yamagishi
2016). It is true that the expression of MYB12 is very low, but MYB12 is lowly expressed in all the
cultivars when compared to other genes. It suggests that it would probably be sufficient to induce the
late stage genes (although F3’H and DFR would have to be introduced first) in anthocyanin
biosynthesis, which might result in at least slight tepal coloration. To summarize, despite the presence
and expression of all necessary transcription factors the cultivar is still white. This is why the most
likely cause are two missing structural genes. However in order to confirm this an introduction of both
structural genes into ‘Lincoln’s tepals should be performed and the phenotype of transformed flowers
evaluated.
51
7
CONCLUSIONS
1. ‘Lincoln’ is missing two structural genes (F3’H and DFR) and bHLH2 transcription factor.
2. The lack of two structural genes in ‘Lincoln’ is the most probable cause of its white flower
colour.
3. Functionality of DFR in ‘Rialto’ is partially questionable due to its different 3D structure.
Together with a mutation in MYB12 (found by Suzuki et al. (2015)) it is the likely cause of
white colouration of ‘Rialto’ flowers.
4. All structural genes in ‘Gran Tourismo’ display much higher expression than all the other
cultivars in this study.
5. No clear link between the expression of bHLH2 and early genes could be found.
6. Commonly used reference genes show a wide gene expression variation among different
stages and cultivars.
52
8
ACKNOWLEDGMENTS
I would like to thank Nur Fatihah and dr. Frans Krens for their guidance and supervision, dr. ing. Jan
Schaart for his help with molecular aspects of this project, especially in relation to the “blue
experiment”. I would also like to thank Marian Oortwijn for her guidance in the laboratory, Robert van
Loo for his help with statistics and all the other people from Plant Breeding department who helped
me during this thesis.
53
9
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56
APPENDIX I CTAB MINI RNA-ISOLATION PROTOCOL
CTAB buffer
1.4 M NaCl
2% CTAB
100 mM Tris pH8.0
20 mM EDTA pH 8.0
2% PVP40
1% beta mercaptoethanol: add just before use !!
Other solutions
*isopropanol
*Chloroform
*8 m LiCL; 33.9 gram/100 ml
*80% EtOH
1. Remove tissue from -70C freezer to liquid N2. Add the plant material to the liquid N2 in the
mortar, and start grinding the tissue just before the N2 has evaporated. Continue grinding until a
very fine powder remains.
2. Transfer 100 mg powder with a cooled spatula into a 2 ml tube and add 750 µl pre-warmed 2%
CTAB buffer, vortex well and incubate 20 min at 60∘C and shake once a while.
3. Add 750 µl chloroform and vortex well.
4. Spin at 12000 rpm for 5 minutes.
5. Transfer top phase (600 µl) to new 1.5 ml tube and add 500µl isopropanol.
6. Invert tubes gently (you may see a precipitate of nucleic acids (DNA+RNA))
7. Spin 5 min at 12000rpm
8. Carefully pour of supernatant and add 500 µl 70% EtOH
9. Spin for 5 min at 12000rpm.
10. Resuspend pellet (do not dry) in 150 µl MQ
11. Add 50µl of 8 M LiCl and incubate o/n at 4C or 30 min at -20∘C.
12. Spin 30 minutes at 14000 rpm at 4C.
13. Wash pellet with 80% EtOH and spin again for 5 minutes.
14. Dry pellet briefly and dissolve pellet in 25 l water.
57
APPENDIX II DNASE TREATMENT AND CDNA SYNTHESIS
DNAse treatment
50x
…μl RNA (=1μg)
1μl 10xDnase I reaction buffer 50
1 μl Dnase I
50
… μl MQ
Vt = 10μl
2μl per well
15 minutes RT (20oC)
Add 1μl EDTA (25mM)
10 minutes 65oC
cDNA synthesis with iScript cDNA synthese kit from Biorad
50x
11μl RNA (Dnase treated)
4 μl 5x iScript reaction Mix
1 μl iScript reverse transcriptase
4 μl Rnase free water
Vt = 20 μl
55
200
50
200
Program in the PCR machine
5 minutes 25oC
30 minutes 42oC
5 minutes 85oC
5 minutes 4oC
5 minutes 85oC
Hold at 10oC
58
APPENDIX III PRIMER LIST
List of all the primers used in this study
Gene
Forward primer
Reverse primer
Reference
RT-qPCR
Reference genes
LhUBQ4
GGTATCCCTCCGGACCAG
ATGGTGTCCGAACTCTCCAC
Yamagishi
2011
LhActin
ATGTATGTTGCAATCCAGGCTGTGC
ATACCAGTAGCTTCCATTCCAACCA
Yamagishi et
al., 2010
LhGAPDH
CTACTGTGCACGCCATCACT
ACACATCGACAGTGGGAACA
-
Structural genes
LhbHLH2
GGCCAAGCAACTCAAAAGAG
CAACTCGATATGGGCACCTT
-
LhMYB12
GGGTGAAGCTGAACCAAAAA
GTCCCATTGGAGAATTGCAT
-
LrMYB15
CTCTTGGGAAACAGGTGGTC
TCGACATGGCCGGTTTTCTG
-
LhCHSa
TGGGACTCACCTTCCATCTC
CATGTTTCCGTACTCGCTCA
-
LhCHSb
CTGAAGCTGGCGCTGGACAAAAAG
GGTAGTGATCGGAATGCTGTGAAGA
LhCHIa
TCCATCCTCTTCACCCAGTC
CCTTGAGAAGCTCGGAAATG
Suzuki et al,
2015
LhCHIb
GCGGTCGATAAGTACGAGGA
TCCCACCCAAATACCACTTC
-
LhDFR
ATATGCCATCCCCCAAAAGT
GCAACCCAAATCCAGTTCAT
Yamagishi
2011
LhF3H
TGCCTTTGTTGTCAATCTCG
GCATCCAAAACTGGCTTCTC
-
LhF3’H
ACGCACGACACAAACTTCAG
CGAGTGCTTTCTGGGAAAAG
Suzuki et
al.,2015
AlF3’H2
ACGCACGACACAAACTTCAG
CGAGTGCTTTCTGGGAAAAG
LhF3’H3
CCTCCACCAAGCTCAGAAAG
TGGCGTTCTTAGGAATTTGG
-
LhF3’H4
TTCACAGCAGGGACAGACAC
CATTGATGGTGCAAGATTCG
-
LhANS
GGGGGAATGGATGACCTACT
GTTGGTGAGGAGGAAGGTGA
Suzuki et al.,
2015
Yamagishi
2011
Long fragments
LsANS_1062
ACCGAGATCATGCCGTTG
GTCCTCCTCTGTCTTCTTGA
Suzuki, 2015
LsCHIa_683
AGCTCCGAAGCTGGAGGTC
CACTAACAATGGTAGGCTCTTCCT
Suzuki, 2015
59
LsCHSb_1178
CAGTCCAACCATGTCGAAGA
TCGGAAGGCTGTGAAGAACT
Suzuki, 2015
LhF3H940
CGACTACCTTCCTCCCAACA
CAACTTCGGTGGGTTTCTTC
-
LsDFR_447
CCAGCCCACAATAAATGGAG
CCACTCGCTTCTGGATTCTC
-
LhDFR_628
CATGGGATTTTGCAAAGGAG
TGGTCCTCCAAAGCTTACTGA
-
LsMYB12_full
TTTCAAACGTTTATTGCCTCCGC
CAACTTCGGAATCACTCCAAAG
-
LhMYB12full2
GCTTCAACGAGGATGAGGAG
TCATTTGGCGGAAAAACTCT
-
LhMYB15_60
0
CATCCCAGTTAGAATGCAGAAA
AAATTATACCAACGTTTGACTGTTCC
-
LsbHLH2_600
CACTCAACCTCCAACCCAGT
TGCTGTTTGCCAAGAATGAG
-
LhbHLH2_811
AGGCACACTGCAGGAAGAAC
TACCATCATCACCGACGAGA
-
LhGAPDH_43
1
CTACTGTGCACGCCATCACT
GCCCCATTCATTGTCATACC
-
Blue cloning
DgDFR
gcgtatATGACTGTAGAAACTGTTTGTGTC
A
agatctTCAGTTGCTCCATTTTGTAGCTGA
C
-
DgF3’5’H1
gagtctATGTCTACAAGCTTGTTGCTTGCT
G
agatctCTCCTCAGACTACATATGCAGAG
GGC
-
DgF3’5’H2
ccatggATGCACCTTCACCATGTCTATAAG
C
ggtaccGTCCTCAGACTACATATGCAGAG
GGT
-
Primer name starting with Lh, Ls, Lr or Dg means the primers were designed based on the hybrid lily, L.
Speciosum, L. regale or D. grandiflorum sequences. Prefix Al indicates a primer pair designed based on
an alignment of several sequences. Primers without indicated reference were designed for the
purpose of this study.
60
APPENDIX IV QPCR AND PHUSION PROTOCOLS
RT-qPCR protocol
40x
95°C
95°C
60°C
02:00min
00:10min
00:25min
95°C
10:00min
65°C
65°C
0:05min
0:05/cycle
Phusion PCR protocol
98°C 00:30min
35x 98°C 00:10min
57°C 00:30min
72°C 00:55min
72°C 10:00min
10°C ∞
61
APPENDIX V MICROSPIN™ G-50 COLUMNS PROTOCOL
Recommended usage.
Column Preparation:
1.
2.
3.
4.
Resuspend the resin in the column by vortexing gently.
2. Loosen the cap one-fourth turn and snap off the bottom closure.
Place the column in a 1,5ml screw-cap mep for support.
Pre-spin the column for 1 minute 3000rpm. Start timer and centrifuge simultaneously.
Sample Application:
5. Place the column in a new 1,5ml screw-cap tube and slowly apply the sample (max 50µl) to
the center of the angled surface of the resin. Do not disturb resin! Careful application of the
sample to the centre of the bed is essential for good separation. Do not allow any of the
sample to flow around the sides of the bed.
6. Spin the column for 2 minutes at 3000rpm. The purified sample is collected in the bottom of
the support tube.
7. Discard the column.
62
APPENDIX VI PROTEIN SEQUENCE ALIGNMENT
63
64
65
Red rectangular in the DFR alignment is the conserved binding site of NAPDH (Bashandy et al. 2015).
66
67
APPENDIX VII POLYMORPHIC SNPS IN CHIa GENE
68
APPENDIX VIII „BLUE EXPERIMENT”
METHODOLOGY
Two genes were chosen for this experiment, F3’5’H and DFR. An attempt was made to isolate two
different alleles of F3’5’H as an alignment of the sequences obtained from the GenBank database
indicated a presence of two alleles. Firstly the long primers were designed with restriction sites at the
ends of the sequences (see Appendix III). Following the advise of dr. Jan Schaart the primers were
used to amplify the product which was later introduced into an intermediate cloning vector pCRII in
TOPO® cells from Invitrogen (for a protocol see Appendix IX). Transformed cells were first grown on
the solid selective LB medium with 50mg/l of kanamycin. After an overnight grow in 37°C, six colonies
per sample were chosen. Based on sequence data (with use of M13 primers) one sample per each
gene/allele was chosen to continue with. The intermediate vector was digested with appropriate
restriction enzymes (depending which restriction sites were present in the PCR product, see protocol
in Appendix X) and the insert was ligated with pRAPAM vector (see protocol Appendix XI). Additionally
DFR and two alleles of F3'5'H PCR products were directly ligated with pRAPAM vector, without
introducing them into pCRII vector in TOPO cells.
Afterwards pRAPAM vectors with an insert (originating from TOPO cells and the enzymatic reaction)
were transformed into the 25µl of Golden cells per transformation, following the manufacteurs
protocol (with a minor change, only 1µl of β-mercaptoethanol was added instead of 4µl). Golden cells
were grown overnight on the selective LB medium with 50mg/l of ampicillin. As the colonies were
small and many, 12 colonies per each plate were chosen and left for growth for an hour in 300ul LB
media with ampicilin. Afterwards a colony PCR was performed and based on the results from the gel
electrophoresis it was decided to grow overnight one colony carrying the DFR gene and 7 colonies
carrying an F3’5’H gene in 3ml liquid LB media with 50mg/l ampicilin. Then part of the colony was used
to make a glycerol stock, while the rest was used for plasmid isolation. Plasmid samples were send for
sequencing. Two samples of the same allele from F3’5’H and one sample from DFR had good quality
reads overlapping with the reference genes of interest.
For further research plans, the inserts from the three successful samples will be ligated into a binary
vector pBINPLUS and that vector will be transformed into Agrobacterium tumerfaciens, which will be
later used for lily flowers transformation. It would be interesting to transform pink ‘Perth’ and white
‘Rialto’, because in ‘Perth’ it is known that the pathway is functional, and the addition of F3’5’H gene
itself should already shift the pigment production towards blue. An addition of DFR is expected to
intensify this effect. In ‘Rialto’ the hypothesis is that due to an impaired function of DFR and possibly
MYB12 the colour is not developed. It is expected that an overexpression of DFR could induce some
pigment production (in combination with F3’5’H a pigment production from the blue branch of the
anthocyanin biosynthesis pathway). If this would not show to be successful perhaps an additional
transformation with MYB12 gene originating from one of the coloured cultivars (e.g. ‘Gran Tourismo’
or ‘Perth’) could induce the pigment production.
69
APPENDIX IX TOPO® PROTOCOL
1µl of purified PCR product
0.5 µl pCRII vector
0.5 µl salt solution
1 µl miliQ
Final volume 3 µl
Leave for 30min in room temperature
Transfer 2 µl to TOPO® cells
70
APPENDIX XDIGESTION PROTOCOL
Double Digestion with SacI, BglII
We recommend: The first digestion should be performed in 1X Tango buffer (low salt concentration buffer) with 2-fold excess
of SacI. Incubate at 37°C for 1 hour.
When the first digestion is complete, add 10X concentrated Tango buffer (amount "V") to a final 2X concentration (high salt
concentration buffer) and BglII.
V=A/8, A - the starting volume of the reaction mixture.
Incubate at 37°C for 1 hour.
DFR
19 µl of purified DFR PCR product
5 µl of Tango restriction buffer (=1X)
2 µl of SacI (=SstI)
24 µl MQ
50 µl total volume; digest for 1h at 37°C
add 6.25 µl of Tango restriction buffer (=2x)
1 µl of BglII
digest for another 1h at 37°C
F3’5’H
19 µl of purified F3’5’H PCR product
10 µl of Tango restriction buffer (=2x!)
1 µl of NcoI
1 µl of BglII
19 µl MQ
50 µl total volume; digest for 1h at 37°C
pRAPAM for DFR
10 µl pRAPAM vector (1 µg)
5 µl of Tango restriction buffer (=1X)
2 µl of SacI (=SstI)
33 µl MQ
50 µl total volume; digest for 1h at 37°C
add 6.25 µl of Tango restriction buffer (=2x)
71
1 µl of BglII
digest for another 1h at 37°C
pRAPAM for F3’5’H
10 µl pRAPAM vector (1 µg)
10 µl of Tango restriction buffer (=2x!)
1 µl of NcoI
1 µl of BglII
28µl MQ
50 µl total volume; digest for 1h at 37°C
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APPENDIX XI LIGATION PROTOCOL
vector:insert ratio = ~1:3; DFR=1.0 Kb; F3’5’H=1.7 Kb; pRAPAM=2.7 Kb
total vector + insert = 100 ng DNA
in 10 µl final volume
for DFR: 50 ng DFR-insert + 50 ng pRAPAM-vector
for F3’5’H: 65 ng F3’5’H-insert + 35 ng pRAPAM-vector
ligation (set up in 500 µl Eppendorf tubes)
x µl pRAPAM vector
Y µl DFR, F3’5’H insert (in case amount of available insert is limited, use as much as possible)
1 µl T4-ligase reaction buffer (10X); Fermentas
0.2 µl T4 ligase (5U/µl)
Z µl MQ
10 µl final volume
incubate 1h at 22 °C in old PE-thermocycler
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