Breeding of polyploid heather (Calluna vulgaris)

Breeding of polyploid heather (Calluna vulgaris)

Annett Przybyla • Anne Behrend •

Christian Bornhake • Annette Hohe

Received: 11 February 2014 / Accepted: 11 April 2014

� Springer Science+Business Media Dordrecht 2014

Abstract Calluna vulgaris is an important land-

scaping plant in Northern Europe. As C. vulgaris is the

only species within the genus Calluna, the available

gene-pool for breeding is rather narrow and pheno-

typic variation is limited. Hence, a breeding program

for polyploids was set up in order to broaden

phenotypic variation in this important ornamental

crop. Therefor basic genetic characteristics of poly-

ploid C. vulgaris were analyzed using the progeny of a

spontaneously occurring tetraploid genotype. With the

help of morphological and molecular marker analysis,

it was determined that the autotetraploid plant prob-

ably arose from unreduced gametes resulting from first

division restitution. Tetraploids displayed tetrasomic

inheritance with free combination of homologous and

homeologous chromosomes. Triploids were semi-

fertile with a high rate of aneuploids in their progeny.

As the so-called bud-blooming flower type is a trait of

outstanding interest in breeding of C. vulgaris, a

strategy for efficient breeding of triploid bud-bloomers

was deduced.

Keywords AFLP � Aneuploids � First division

restitution � Tetrasomic inheritance � Triploid

fertility

Introduction

Heather (Calluna vulgaris) is a bedding and landscap-

ing plant of considerable importance for the autumn

season in Northern Europe. During the past two

decades, breeding activities concentrated on so-called

bud-blooming genotypes which display outstanding

longevity of the flowers (Borchert et al. 2009, 2012).

However, the gene-pool available for breeding is

rather narrow, because C. vulgaris is the only species

within the genus Calluna. A broad fingerprinting

analysis of 74 C. vulgaris genotypes revealed a close

relationship between cultivars and wild-types from

different provenances (Borchert et al. 2008). Hence,

the development of polyploid cultivars with larger

organ size, especially larger flowers, and an overall

stronger habitus compared to diploids is an attractive

opportunity for broadening the phenotypic variation in

this ornamental plant.

Breeding of polyploids is more demanding com-

pared to diploids (Acquaah 2012). For the develop-

ment of a successful polyploid breeding program, it is

necessary to work out knowledge on the basic genetic

characteristics of the respective plant on polyploid

level. Therefore, the aim of the current study was to

A. Przybyla � A. Behrend � A. Hohe (&)

Leibniz-Institute of Vegetable and Ornamental Crops,

Kuehnhaeuser Strasse 101, 99090 Erfurt, Germany

e-mail: [email protected]

C. Bornhake

Heidepflanzen de Winkel, Douvenberg 34,

47574 Goch-Kessel, Germany

123

Euphytica

DOI 10.1007/s10681-014-1117-1

analyze the origin of polyploids, the inheritance of the

important trait ‘‘flower type’’ on polyploid level and

the fertility of plants with different ploidy levels with a

focus on triploids. Analyses were performed by

characterization of the progeny of a spontaneously

occurring tetraploid genotype using morphological

and molecular marker analyses. Due to the extremely

small size of chromosomes in C. vulgaris (DNA

content: 1.18/2C, 2n = 2x = 16 (Borchert et al. 2009;

Behrend et al. 2013)), no cytological methods have

been included in the study. C. vulgaris belongs to the

family Ericaceae as does Vaccinium, a genus about

which considerable knowledge on breeding of polyp-

loids exists (Lyrene et al. 2003). Hence, the results of

the current study are mainly discussed on the back-

ground of comparative studies in Vaccinium.

Materials and methods

Plant material

The tetraploid plant 7705 occurred spontaneously in

the progeny of a crossing of the diploid cultivars

‘Maria’ and ‘Perestroijka’. It was left within a field of

diploid plants for open pollination. Seeds were

harvested in autumn and sown in the following early

spring. The resulting progeny contained 466 plants

(named population 2008/177).

Other genotypes included in the study were diploid

cultivars, diploid wild-types from different prove-

nances and a triploid genotype that occurred sponta-

neously in the offspring of a cross of the diploid

genotypes ‘Maria’ and ‘Boskoop’.

Morphological characterization

All plants of population 2008/177 were subjected to

morphological analysis with regard to leaf size, flower

size, flower type and flower density. ‘Flower type’ was

categorized as ‘wild-type’ or ‘bud-bloomer’. Leaf and

flower size were classified as ‘small’, ‘medium’ and

‘large’. Likewise, flower density was classified as

‘loose’, ‘medium’ and ‘compact’.

For detailed quantitative analysis of organ size, leaf

size was chosen as a representative parameter. Ten

diploid genotypes were analyzed as a control group. In

addition, the only four diploid genotypes of progeny

2008/177 as well as ten selected triploid and ten

selected tetraploid genotypes of this population were

included in the analysis (Table 1). To determine the

length and width of the leaves, the first three leaves of

a short-shoot were taken from three plants per

genotype. The leaf length and width were measured

with the help of a binocular microscope (Olympus

SZX10), camera (Olympus DP26) and imaging soft-

ware (Olympus cellSens).

Flow cytometric analysis

Plants were screened for their ploidy level using flow

cytometry (CyFlow� space, Partec) with Petu-

nia 9 hybrida (cv. ‘Mitchell’) as internal standard.

For ploidy analysis of progeny 2008/177, the CyStain

PI absolute P reagent kit (propidium iodide staining,

Partec) was used according to the manufacturer’s

instructions. About 0.5 cm2 leaf tissue of Calluna and

Petunia (reference type for internal standardization)

was chopped using a razor blade in 500 ll extraction

buffer for 30–60 s. After 30–90 s of incubation, the

sample was filtered through a Partec 50 lm CellTrics

disposable filter. 2.0 ml staining solution (containing

12 ll Propidium iodide and 6 ll RNAse stock solu-

tion) with propidium iodide was added to the test tube.

The samples were incubated for 30–60 min protected

from light and subsequently analyzed (at least 3,000

particles). The ratio of peak positions of Calluna and

Petunia was used for ploidy determination (Fig. 1).

The offspring of five triploid genotypes from

population 2008/177 was screened using the CyStain

UV precise P reagent kit (DAPI staining, Partec)

according to the manufacturer’s instructions. 0.5 cm2

leaf tissue of Calluna and Petunia was chopped in

400 ll extraction buffer. After 30 s up to 5 min of

incubation the sample was filtered (Partec 50 lm

CellTrics disposable filter). 1.6 ml staining solution

was added to the test tube and finally the samples were

analyzed (at least 2,000 particles).

AFLP analysis

The parental cultivars ‘Maria’ and ‘Perestroijka’, their

tetraploid offspring ‘7705’ as well as selected plants of

progeny 2008/177 were subjected to AFLP analysis

(Vos et al. 1995). As non-related control genotypes the

cultivar ‘Hammondii rubrifolia’, and wild-types from

Germany, Tasmania and Russia were included in the

analysis.

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The AFLP procedure was conducted and its repro-

ducibility tested according to Borchert and Gawenda

(2010) with adapter (Table 2) and primer sequences

from Behrend et al. (2013). Ten primer pairs were

chosen for analysis (Table 3).

Results

Characterization of the progeny of the tetraploid

plant 7705

The tetraploid F1 plant 7705 was left for open

pollination in a breeding field of several thousand

diploid heather plants. This field did only contain one

other polyploid plant, a triploid. Seeds were harvested

and germinated resulting in a progeny of 466 plants

(named 2008/177 in the following). Flow-cytometric

analysis (Fig. 1) was used for determination of the

ploidy level. 431 plants (92.5 %) were tetraploid, 31

plants (6.7 %) were triploid and four plants (0.9 %)

were diploid. Two triploid plants and 74 tetraploid

plants did not flower, for two tetraploids only the

flower type, but no other flower characteristics could

be determined.

For analysis of the relation between organ size and

ploidy level, leaf size (first three leaves of a short-

shoot) has been chosen as the best representative

measure, as in case of flowers the flower type (wild-

type or bud-bloomer) might influence the flower size.

Ten diploid cultivars and genotypes of diverse genetic

background were used as a control and compared to

the four diploid genotypes from progeny 2008/177, to

nine randomly chosen triploid and nine randomly

chosen tetraploid genotypes of progeny 2008/177. In

addition to these polyploid genotypes from the prog-

eny 2008/177, a triploid genotype found in another

breeding line and the tetraploid mother plant 7705

were included in this analysis, resulting in at least ten

plants analyzed for each ploidy level (Fig. 2). Overall,

there was a clear correlation between ploidy level and

leave size. The average leaf length was 2.0 mm for

diploids, 2.4 mm for triploids and 2.7 mm for

tetraploids.

On average, polyploid plants, including triploids

and tetraploids, developed an attractive strong habitus

compared to diploids (Fig. 3). For breeding, a com-

bination of large flowers with high flower density is

important. Therefore, the occurrence of this pheno-

typic combination has been analyzed within the

triploid and the tetraploid plants (Table 4). Although

the majority of plants with high flower density

displayed a small flower size, a combination of the

desired traits ‘flower density’ and ‘flower size’

occurred in 3.4 % (1 plant) of the triploids and in

2.8 % (10 plants) of the tetraploids.

The origin of the triploid and especially of the

diploid genotypes in progeny 2008/177 was unclear.

Therefore, the tetraploid genotype 7705, all surviving

diploid and triploid genotypes and 45 tetraploid

genotypes of its progeny as well as three diploid

genotypes from natural origin and one diploid cultivar

with a severely differing phenotype (‘Hammondii

rubrifolia’) were subjected to an AFLP-analysis. The

use of ten different primer combinations resulted in a

sum of 180 distinguishable AFLP loci and an average

Table 1 Calluna vulgaris genotypes included in the morphological comparison of plants with different ploidy level

Diploid control group Diploid Triploid Tetraploid

‘Alicia’ (bb) 2008/177/15 (wt) 2008/177/142 (wt) 2008/177/1 (wt)

‘Athene’ (bb) 2008/177/251 (wt) 2008/177/200 (wt) 2008/177/2 (wt)

‘Maria’ (bb) 2008/177/360 (wt) 2008/177/328 (bb) 2008/177/4 (wt)

‘Mariella’ (bb) 2008/177/455 (wt) 2008/177/330 (bb) 2008/177/14 (wt)

‘Perestroijka’ (wt) 2008/177/359 (wt) 2008/177/21 (wt)

‘Venetia’ (bb) 2008/177/382 (bb) 2008/177/27 (wt)

‘Wink 2-2006’ (bb) 2008/177/392 (wt) 2008/177/55 (wt)

‘Wink 3-2006’ (bb) 2008/177/464 (wt) 2008/177/64 (wt)

Wild-type 1 (Italy, wt) 2008/177/466 (wt) 2008/177/70 (wt)

Wild-type 2 (Germany, wt) Spontaneous triploid from the cross ‘Maria’ 9 ‘Boskoop’ 7705 (wt)

The flower type of the genotypes is given in brackets (bb bud-bloomer, wt wild-type)

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of 104.4 bands per genotype. Genotype 7705 had 106

AFLP markers. For analysis of the origin of single

genotypes in population 2008/177, the number of

AFLP loci of the progeny not present in its mother

plant 7705 was determined. On average, this was only

the case for 8.33 loci of the descendants. However,

these rates differed considerably with regard to the

ploidy level (Fig. 4). Whereas 27.34 % of the bands

present in the diploid control genotypes were absent in

genotype 7705, only 3.74 % of the bands of the

tetraploid genotypes were not present in genotype

7705. As the tetraploid genotypes in the progeny

2008/177 might only have arisen from a selfing of

genotype 7705, all bands present in the progeny should

also occur in the only parent plant. Hence, these

3.74 % represent the experimental error and the

average percentage of ‘‘new’’ bands resulting from

recombinational events that might generate new

amplificates. In contrast, in the diploid and triploid

genotypes of the progeny 12.47 and 10.37 % of the

bands have not been inherited from genotype 7705.

Fig. 1 Flowcytometric analyses (DAPI staining) of diploid (a),

triploid (b) and tetraploid (c) Calluna vulgaris. Ploidy has been

determined by simultaneous measurement with Petunia 9 hyb-

rida (10,000 particles). The ratio of peak positions has been

determined to be 0.37 for diploid genotypes, 0.56 for triploid

genotypes and 0.74 for tetraploid genotypes

Table 2 Adapter sequences (Behrend et al. 2013) used for

AFLP-analysis

MseI adapter 50-GAC GAT GAG TCC TGA G-30 (forward)

50-TAC TCA GGA CTC AT-30 (reverse)

HindIII

adapter

50-CTC GTA GAC TGC GTA CC-30

(forward)

50-AGC TGG TAC GCA GTC TAC-30

(reverse)

Table 3 Sequences of primer pairs (Behrend et al. 2013)

chosen for AFLP-analysis

Primer

pair

MseI primer HindIII primer

1 50-GAT GAG TCC TGA

GTA A ? TCT-3050-GAC TGC GTA CCA

GCT T ? CGA-30


GTA A ? TCT-3050-GAC TGC GTA CCA

GCT T ? AGT-30


GTA A ? TCC-3050-GAC TGC GTA CCA

GCT T ? CAT-30


GTA A ? TCC-3050-GAC TGC GTA CCA

GCT T ? ACA-30


GTA A ? TCG-3050-GAC TGC GTA CCA

GCT T ? CA-30


GTA A ? TCG-3050-GAC TGC GTA CCA

GCT T ? AC-30


GTA A ? TAC-3050-GAC TGC GTA CCA

GCT T ? CA-30


GTA A ? TAC-3050-GAC TGC GTA CCA

GCT T ? AC-30


GTA A ? AGT-3050-GAC TGC GTA CCA

GCT T ? CGA-30


GTA A ? AGT-3050-GAC TGC GTA CCA

GCT T ? AGT-30

The selective nucleotides are given in italics

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However, in each ploidy group at least one plant

displayed no deviating AFLP band compared to

genotype 7705.

Origin of the tetraploid genotype 7705

The genotype 7705 has been found in the F1-progeny

of a cross between ‘Maria’ and ‘Perestroijka’, but it is

not known, whether it resulted from somatic doubling

or from the formation of unreduced gametes. Somatic

doubling can result only in the genetic constitutions

AAAA, AAaa and aaaa (by doubling of the diploid

genotypes AA, Aa and aa). The same is true for

genotypes that arose from the union of two unreduced

gametes resulting from second division restitution

(possible recombination events are neglected). In

contrast, the formation of a zygote from unreduced

gametes originating from first division restitution can

also result in the genotypes AAAa and Aaaa, with

Aaaa resulting in a specific segregation ratio in the

offspring. Therefore, all 106 AFLP loci of genotype

7705 were subjected to a segregation analysis in the 45

randomly chosen tetraploid genotypes of its offspring

(Fig. 5). From this, possible genetic constitutions of

these loci in the tetraploid plant 7705 according to a

V2-test have been deduced assuming tetrasomic

inheritance. 13.21 % displayed odd segregation ratios.

71 loci did not segregate at all which could result from

the genotypes AAAA, AAAa and AAaa. 10 loci

(9.43 %) displayed a segregation ratio that can only

result from an Aaaa constitution in the genotype 7705.

Fig. 2 Organ size of leaves of Calluna vulgaris from progeny

2008/177 with different ploidy levels (diploid: n = 4, triploid:

n = 10, tetraploid: n = 10). As control, characteristics of ten

different diploid cultivars have been determined

Fig. 3 Flowering shoots of the tetraploid plant 7705 (a) and of three selected plants of its progeny 2008/177, each representing one

ploidy level (b diploid, genotype no. 360; c triploid, genotype no. 124, d tetraploid, genotype no. 9)

Table 4 Flower morphology of polyploid Calluna vulgaris

genotypes from progeny 2008/177 (absolute number of plants

and percentage with regard to the ploidy level)

Flower density Flower size

Small Medium Large

29 Triploid genotypes

Loose 0 (0.0 %) 7 (24.1 %) 0 (0.0 %)

Medium 1 (3.4 %) 5 (17.2 %) 0 (0.0 %)

Compact 9 (31.0 %) 6 (20.7 %) 1 (3.4 %)

355 Tetraploid genotypes

Loose 12 (3.4 %) 92 (25.1 %) 19 (5.4 %)

Medium 13 (3.7 %) 82 (23.1 %) 19 (5.4 %)

Compact 28 (7.9 %) 80 (22.5 %) 10 (2.8 %)

A combination of large flowers with high flower density is

desired for breeding (bold letters)

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Inheritance of the trait ‘‘flower type’’ on tetraploid

level

The trait of utmost interest in breeding of heather is the

flower type. Here, in the progeny 2008/177 five out of

the flowering triploid plants (17.2 %) and 16 out of the

flowering tetraploid plants (4.5 %) were bud-bloom-

ers. Due to the open pollination of the genotype 7705,

the triploid plants probably resulted from fertilization

with pollen from diploid plants of unknown origin.

Therefore, a segregation analysis can only be done for

the tetraploid genotypes which—with a very high

probability—resulted from selfing of the tetraploid

mother plant 7705 (although few cross pollinations

with unreduced pollen from diploid plants cannot be

excluded). In diploid C. vulgaris, the flower type is

determined by one locus, wild-type flowers are

dominant over bud-bloomers (Borchert and Hohe

2009). Using the V2-test, it was analyzed, whether the

segregation ratio of 341:16 (wild-type:bud-bloomers)

of the tetraploid genotypes might match a 35:1

segregation resulting from a tetrasomic inheritance

with four recessive alleles being necessary for the bud

flowering phenotype. As the resulting V2-test statistic

(3.838) was lower than the critical value of 3.841, the

hypothesis was not rejected. On the other hand, also

the possibility of disomic inheritance has been tested.

Depending on the constitution of genotype 7705 (Aa

Aa or AA aa) this might result in a segregation of 15:1

(Aa Aa) or in fixed heterozygosity (AA aa). As 16 bud-

Fig. 4 AFLP analysis of progeny 2008/177. Percentage of

AFLP loci differing between the seed bearer 7705 and its

offspring with different ploidy level (mean ? SD). As a control,

four diploid plants with diverse genetic background compared to

genotype 7705 were analyzed

Fig. 5 Segregation analysis

of 106 AFLP loci from

genotype 7705 in 45

tetraploid plants of its

progeny 2008/177. Absolute

numbers of AFLP loci

showing the respective

segregation ratio are

presented. Using a V2-test

all given segregation ratios

have been compared to

expected ratios resulting

from the genetic constitution

of a locus in genotype 7705

(tetrasomic inheritance

assumed)

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bloomers occurred in the progeny, fixed heterozygos-

ity could be ruled out. However, a 15:1 segregation

was not rejected (V2-test statistic: 1.905).

Fertility of triploid offspring

It has been expected that triploid plants suffered from a

reduced fertility due to impaired meiosis. However,

after open pollination triploid plants displayed con-

siderable seed set. From five different genotypes

altogether 346 seeds have been harvested from which

117 seedlings were obtained. Germination rate and

growth of seedlings was low compared to standard

diploid progenies. As soon as the seedlings had

reached a sufficient size, their ploidy level has been

determined using flow-cytometry (Table 5). More

than half of the plantlets were diploid, 27 % were

aneuploid, 13 % tetraploid and 3 % triploid. From the

aneuploid plants, the majority was hyperdiploid.

Discussion

Due to their attractive morphology, polyploid C.

vulgaris might be of interest for breeders and growers

of heather, since the phenotypic and genetic variability

is very limited among diploid C. vulgaris (Borchert

et al. 2008). Therefore, we analyzed basic genetic

characteristics of polyploid C. vulgaris that are

necessary for the development of breeding strategies.

This analysis was based on a progeny of 466 plants

(named progeny 2008/177) that has been obtained by

open pollination of the tetraploid plant 7705 which

arose spontaneously in the F1-progeny of a cross

between a diploid bud-bloomer and a diploid pollina-

tor with wild-type flower morphology.

Characterization of the progeny of the tetraploid

plant 7705

Although the seed bearing tetraploid genotype 7705

has been left for open pollination within a field of its

diploid sister plants, more than 90 % of its offspring

were tetraploid. C. vulgaris has been classified as

being mixed mating with predominant allogamy

(Mahy and Jacquemart 1998). Hence, a predominantly

triploid progeny originating from pollination of the

tetraploid seed bearer with diploid pollen has been

expected. The tetraploid offspring can only have

resulted from selfing (neglecting the rare possibility of

fertilization with unreduced pollen from diploid

plants) which is not the favorite mating system in C.

vulgaris. Therefore, it is assumed that a considerable

triploid block exists in C. vulgaris that impedes

triploid formation due to malfunction of the endo-

sperm (Kohler et al. 2010). Moreover, enhanced rates

of self-fertilization in polyploids compared to their

diploid relatives are frequently observed (Barringer

2007). The presumption of a triploid block in C.

vulgaris is also supported by observations of the

Table 5 Ploidy analysis of the progeny of triploid genotypes of Calluna vulgaris (open pollination)

Genotype 5 108 204 299 374 Sum Percentage

Number of seeds 100 143 29 70 4 346

Number of seedlings 39 43 6 26 3 117

Germination rate (%) 39 30 21 37 75

Diploid 27 30 1 8 66 56

Triploid 1 1 2 4 3

Tetraploid 4 3 8 15 13

Euploid 32 34 1 18 0 85 73

Hypo-diploid 2 2 2

Hyper-diploid 6 6 2 2 16 14

Hypo-triploid 1 1 2 2

Hyper-triploid 3 3 4 10 9

Hypo-tetraploid 1 1 2 2

Hyper-tetraploid 0 0

Aneuploid 7 9 5 8 3 32 27

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occurrence of spontaneous polyploids in the breeding

field: Tetraploids probably arise by the random

coincidence of an unreduced egg cell with unreduced

pollen. Consequently, triploids—whose emergence

requires the formation of only one unreduced gam-

ete—should occur much more often in comparison to

tetraploids. However, the spontaneous formation of a

triploid plant has been observed only once in the

progeny of a cross between the cultivars ‘Maria’ and

‘Boskoop’. In the genus Vaccinium that is also

belonging to the Ericaceae family, a strong but not

complete triploid block has been reported (Dweikat

and Lyrene 1988; Lyrene et al. 2003). However, in this

genus, heteroploid crosses usually are also interspe-

cific crosses, which might likewise impair fertilization

success.

The origin of the triploid and especially of the

diploid plants within the progeny 2008/177 remained

unclear. Triploids might have arisen by pollination of

the tetraploid genotype 7705 with the 1n = 1x pollen

cloud of the breeding field. On the other hand, triploids

might also have resulted from pollination of 1n = 2x

egg cells with 1n = 1x pollen of genotype 7705 itself,

originating from irregular meiosis which might occur,

as this genotype is an autopolyploid. Likewise,

diploids might simply be contaminants or they

resulted from pollination of irregular 1n = 1x female

gametes of genotype 7705 with pollen from diploid

plants in the breeding field. Generation of 1n = 1x

pollen by the tetraploid genotype 7705 is the more

improbable alternative, however, one diploid plant

and one triploid plant within its progeny displayed

only AFLP loci that were also present in genotype

7705 itself. Hence, these genotypes might be diploid

and triploid selfing descendants. The overall high

congruence between AFLP loci in genotype 7705 and

non-related diploid cultivars (27.34 %) can be

explained by the low genetic variability in C. vulgaris

(Borchert et al. 2008). On average, the rate of

deviating AFLP loci of triploid offspring was almost

three times higher compared to tetraploid descendants

that were most probably selfings. This is regarded to be

the result of crossing with the pollen of diploid plants

in the breeding field.

Origin of the tetraploid genotype 7705

The tetraploid genotype 7705 itself might have

originated from somatic doubling or from the union

of unreduced gametes resulting from first or second

division restitution. In case of somatic doubling and

second division restitution (neglecting recombination

events) at any locus only the genetic constitutions

AAAA, AAaa and aaaa might result, whereas the

genetic constitutions of AAAa and Aaaa might only

have resulted from unreduced gametes with first

division restitution. The segregation analysis of 106

AFLP loci in 45 tetraploid plants of progeny 2008/177

showed that 9.43 % displayed a segregation ratio that

can only result from an Aaaa constitution in the

genotype 7705. Hence, it is presumed that the

spontaneous incidence of the tetraploid genotype

7705 resulted from unreduced gametes originating

from first division restitution. Generally, it is pre-

sumed that non-reduction of gametes is the major

mechanism of polyploid formation in plants (Ramsey

and Schemske 1998). For the related genus Vaccinium,

it has been reported that unreduced pollen develops

frequently in different diploid species (Ortiz et al.

1992a). Likewise, first division restitution is regarded

to be the origin of unreduced gametes (Ortiz et al.

1992b).

Inheritance of the trait ‘‘flower type’’ on tetraploid

level

A trait of superior interest in breeding of C. vulgaris is

the flower type (Borchert and Hohe 2010). So called

bud-blooming genotypes are preferred due to longev-

ity of the flowers. In diploid genotypes, a monogenic

recessive inheritance of this trait has been proven

(Borchert and Hohe 2009). The tetraploid genotype

7705 resulted from the cross between a bud-bloomer

and a genotype with wild-type flower morphology,

both being homozygous with respect to the flower type

locus (wild-type: AA, bud-bloomer: aa). Hence, the

tetraploid offspring 7705 should bear the genetic

constitution AAaa at the flower type locus, irrespec-

tive of its origin through unreduced gametes or

somatic doubling. Therefore, segregation analysis of

its progeny allows to draw conclusions on tetrasomic

or disomic inheritance. In case of tetrasomic inheri-

tance, a segregation ratio of 35:1 (wild-type:bud-

bloomers) is expected. In case of disomic inheritance,

either fixed heterozygosity (i.e. no phenotypic segre-

gation with regard to the flower type, assuming the

genetic constitution AA aa) or a segregation ratio of

15:1 (assuming the genetic constitution Aa Aa) can

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occur. The latter can be excluded due to the known

origin of genotype 7705 that resulted from a cross of

AA and aa and not from a cross between to hetero-

zygous F1 genotypes (Aa). The null-hypothesis of a

35:1 segregation was not rejected for the actual

segregation ratio of 341:16. Hence, a tetrasomic

inheritance is assumed, although the actual segrega-

tion ratio of 21:1 does not clearly match the expected

35:1 ratio. This bias might be caused by the high rate

(17.1 %) of non-flowering genotypes within the

population 2008/177. On the one hand, tetrasomic

inheritance is to be expected for autotetraploids, on the

other hand, the high fertility of genotype 7705 is

surprising, as autotetraploids often suffer from dis-

turbed meiosis. Again, this resembles Vaccinium. In

this genus, the tetraploid species are assumed to be

autotetraploids with non-preferential bivalent chro-

mosome pairing, resulting in a medium to high fertility

and tetrasomic inheritance (Lyrene et al. 2003).

Fertility of triploid offspring

Triploid genotypes might be infertile due to unbal-

anced chromosome numbers (Ramsey and Schemske

1998), which might be useful for breeders in order to

inhibit the use of their cultivars by competing breeding

companies. In addition, infertility can be desirable for

breeding of non-invasive landscaping plants (True-

blood et al. 2010; Rounsaville et al. 2011). Both aims

might be of interest for C. vulgaris, especially as the

morphology of triploids did not differ clearly from that

of tetraploids. However, triploid genotypes turned out

to be fertile. Although the resulting progeny had an

overall low germination rate, the majority of surviving

seedlings displayed an euploid ploidy level, suggest-

ing a selection for euploidy in gametes or embryos.

Ramsey and Schemske (1998) surveyed 23 studies on

fertility of autotriploid plants and report an average

fertility of 39.2 %. Hence, these authors conclude that,

in contrast to widespread expectations, triploids are

often semi-fertile. As the triploid plants in the current

study were left for open pollination in the breeding

field, they might have been fertilized by pollen from

diploids, triploids and tetraploids. In highbush blue-

berry (V. corymbosum), crosses of triploids and

diploids were very difficult (Vorsa and Ballington

1991). Only one triploid genotype was relatively

fertile in crosses with tetraploids and hexaploids. It

was concluded that triploid V. corymbosum generally

lack fertility due to a very low production of pollen

with numerically balanced chromosome numbers.

Hence, although tetraploid C. vulgaris resemble

tetraploid Vaccinium (a related species also belonging

to the Ericaceae family) with respect to the origin of

polyploids, the fertility and the tetrasomic inheritance

of autotetraploids, these genera seem to differ regard-

ing the genetics of triploids.

Conclusion

Breeding of polyploid C. vulgaris is possible. The

phenotype of triploids resembles that of tetraploids,

hence it is even possible to set up a triploid breeding

program. Bud-blooming genotypes are of superior

interest, however this trait is inherited recessively and

bud-bloomers lack stamen. For a high output of bud-

blooming triploids, it is therefore recommended to

cross tetraploid bud-bloomers with diploid F1 plants

that are heterozygous at the flower type locus. Here, a

progeny with 50 % bud-bloomers is expected. Result-

ing triploids are semifertile with a high rate of

aneuploid offspring. Therefore, the invasive potential

of C. vulgaris used in landscaping is reduced only

relatively. Likewise, the breeding of triploids does not

generate a general breeder’s protection against use of

cultivars by competing breeders.

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Breeding of polyploid heather (Calluna vulgaris)