Biomass yield and heterosis of crosses within and between European winter cultivars of turnip rape (Brassica rapa L.).

Abstract

Because of its high growth rate at low temperatures in early spring, there is renewed interest in Brassica rapa as a winter crop for biomass production in Europe. The available cultivars are not developed for this purpose however. An approach for breeding bioenergy cultivars of B. rapa could be to establish populations from two or more different cultivars with high combining ability. The objective of this study was to evaluate the heterosis for biomass yield in the European winter B. rapa genepool. The genetic variation and heterosis of the biomass parameters: dry matter content, fresh and dry biomass yields were investigated in three cultivars representing different eras of breeding by comparing full-sibs-within and full-sibs-between the cultivars. Field trials were performed at two locations in Germany in 2005-2006. Mean mid-parent heterosis was low with 2.5% in fresh and 3.0% in dry biomass yield in full-sibs-between cultivars. Mean values of individual crosses revealed a higher variation in mid-parent heterosis ranging from 14.6% to -7.5% in fresh biomass yield and from 19.7% to -12.7% in dry biomass yield. The low heterosis observed in hybrids between European winter cultivars can be explained by the low genetic variation between these cultivars as shown earlier with molecular markers. In conclusion, a B. rapa breeding program for biomass production in Europe should not only use European genetic resources, but should also utilize the much wider worldwide variation in this species.

Full text

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PLANT GENETICS • SHORT COMMUNICATION
Biomass yield and heterosis of crosses
within and between European winter cultivars
of turnip rape (Brassica rapa L.)
Atta Ofori & Antje Schierholt & Heiko C. Becker
Received: 13 April 2011 /Revised: 20 September 2011 /Accepted: 24 September 2011 /Published online: 15 October 2011
# The Author(s) 2011. This article is published with open access at Springerlink.com
Abstract Because of its high growth rate at low temper-
atures in early spring, there is renewed interest in Brassica
rapa as a winter crop for biomass production in Europe.
The available cultivars are not developed for this purpose
however. An approach for breeding bioenergy cultivars of
B. rapa could be to establish populations from two or more
different cultivars with high combining ability. The objec-
tive of this study was to evaluate the heterosis for biomass
yield in the European winter B. rapa genepool. The genetic
variation and heterosis of the biomass parameters: dry
matter content, fresh and dry biomass yields were investi-
gated in three cultivars representing different eras of
breeding by comparing full-sibs-within and full-sibs-
between the cultivars. Field trials were performed at two
locations in Germany in 2005–2006. Mean mid-parent
heterosis was low with 2.5% in fresh and 3.0% in dry
biomass yield in full-sibs-between cultivars. Mean values
of individual crosses revealed a higher variation in mid-
parent heterosis ranging from 14.6% to −7.5% in fresh
biomass yield and from 19.7% to −12.7% in dry biomass
yield. The low heterosis observed in hybrids between
European winter cultivars can be explained by the low
genetic variation between these cultivars as shown earlier
with molecular markers. In conclusion, a B. rapa breeding
program for biomass production in Europe should not only
use European genetic resources, but should also utilize the
much wider worldwide variation in this species.
Keywords Genetic variation.Heterosis
Today, Brassica rapa is primarily grown as a spring oilseed
crop in Canada and in some northern European regions.
Whereas its cultivation as winter oilseed crop in Central
Europe has nearly ceased, there is renewed interest in B.
rapa as a winter crop for biomass production because of its
high growth rate at low temperatures in early spring (Ofori
and Becker 2008). If cultivated as a pre-crop, it can be
harvested early in the year before planting the major crop,
such as maize, sorghum or sunflower. B. rapa is of
particular interest among Brassica crops as it has a higher
early biomass than B. napus (unpublished results). For
biomass substrate storage and biogas processing, a high dry
matter content is important, therefore, the major selection
criteria for biomass yield are fresh biomass and dry biomass
yields.
The B. rapa species includes highly diverse morphotypes
with a respective varying utilization as oilseed crop, root and
leaf vegetables or forage plants. Zhao et al. (2005) suggested
two major areas of domestication and respective genepools
in Asia and Europe. B. rapa cultivars exhibit a high genetic
diversity within cultivars (Zhao et al. 2005; Zhao et al.
2009), whereas the genetic variance of crosses between B.
rapa cultivars mainly originated from specific combining
ability (SCA) effects (Ofori and Becker 2008). Cultivars of
B. rapa are self-incompatible (Franklin-Tong and Franklin
2000) and completely cross-pollinated. Sakamoto and Nishio
(2001) discussed the use of the self-incompatibility in F1
hybrid breeding. However, an F1hybrid production, based
A. Schierholt (*):H. C. Becker
Department of Crop Sciences,
Georg August Universität Göttingen,
von Siebold Strasse 8,
37075 Göttingen, Germany
e-mail: aschier@gwdg.de
Present Address:
A. Ofori
Plant Breeding and Genetics Division,
Cocoa Research Institute of Ghana,
Post Office Box 8, New Tafo-Akim, Ghana
J Appl Genetics (2012) 53:31–35
DOI 10.1007/s13353-011-0067-8

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on parental inbred lines, requires a high input into parental
line development, combination testing and F1seed produc-
tion, and a breeding method with a lower input, nevertheless
exploiting heterosis, would be of interest. A simple method
of hybrid production in cross-pollinated species is the
crossing of two populations (Falconer and Mackay 1996).
Considering two parental breeding populations of a
diploid outcrossing species with completely random
mating and in linkage equilibrium, the resulting hybrid
shows a higher degree of heterozygosity compared to the
mean of parental populations and an increase in
heterosis. Schuler et al. (1992) found an average of 18%
mid-parent heterosis in seed yield, in 19 intervarietal and
interpopulation crosses in B. rapa with Canola quality.
Niemelä et al. (2006) reported 18% and 23% heterosis in
seed yield for synthetics and composite hybrids, respec-
tively. Ofori and Becker (2008) showed that heterosis for
biomass yield in B. rapa crosses was low in general, but
up to 30% in the best crossing combinations.
Breeding intensity and investment in the development of
populations from full-sib progenies between different
cultivars are much lower than in F1hybrid breeding. For
a ‘neglected’ crop such as winter B. rapa, this might be a
cost-reduced and effective alternative in the breeding of
cultivars for biomass production. In addition to an earlier
study by Ofori and Becker (2008), in which crosses of a
half diallel were tested for GCA and SCA with respect to
the possible use in F1hybrid breeding, in this experiment,
we compare the biomass yield of three groups of
genotypes: of full-sibs from crosses within three B. rapa
winter cultivars, of full-sibs between these cultivars, and
mixtures of these, to evaluate the genetic variation and
heterosis and to draw conclusions for breeding strategies of
B. rapa for biomass production.
The three B. rapa cultivars in this study were selected as
genetically different in their breeding history and represen-
tative for various eras of European winter B. rapa breeding.
The diploid cultivars Steinacher (S), Rex (R), and Largo (L)
were released in 1954, 1984, and 2002, respectively.
Steinacher, the eldest cultivar, is high in glucosinolates
and erucic acid in the seed oil. Rex, a forage type of B.
rapa, is high in glucosinolates but has zero erucic acid and
was released after the first obvious bottleneck in breeding
history, the introduction of zero erucic acid genetic material
into the B. rapa genepool. Largo has “00” quality, zero
erucic acid combined with low seed glucosinolate content,
and was thus released after the second bottleneck in quality
breeding. Seeds of Steinacher were obtained from the
genebank at IPK Gatersleben, Germany, and were multi-
plied under isolation (Ofori et al. 2008), while seeds of Rex
and Largo were obtained from the plant breeders Norddeut-
sche Pflanzenzucht Hans Georg Lembke KG and SW
Seeds, respectively.
Crosses between plants of the three cultivars (R×R, S×
S, L×L) were performed by isolation of two parental plants
with a bag. Nine crosses of each crossing combination were
harvested at Reinshof experimental station in 2005. The
resulting 27 crossing combinations (R×R, S×S, and L×L)
are referred to as full-sibs-between (FSbetween). Three plants
of each cultivar were selfed to prove their self-
incompatibility and, as expected, showed hardly any seed
set (data not shown).
The seed of full-sibs-within (FSwithin) cultivar crosses
was produced by isolation of two plants each from the same
cultivar. Equal quantities of seed from ten FSwithincrosses
per cultivar (R×R, S×S, and L×L) were bulked for the
field evaluation of FSwithin.
Three mixed bulks (FSmix) of the combinations R×L,
R×S, and L×S were composed of FSwithinand FSbetween,
which are genetically equivalent to a synthetic population
(syn-1 generation) developed by random mating of a
mixture of two cultivars each. Equal quantities of seed
from the nine crosses of each FSbetweencombination were
bulked. The three FSmixbulks were produced by mixing
50% FSbetween bulked seed and 25% of the two
corresponding parental FSwithin lots. The proportions of
the lots were based on their 1,000 seed weight.
The plant material, including the three parental
cultivars, was tested at Göttingen and Einbeck in central
Germany in the growth season 2005–2006. At both
locations, a lattice field design with two replications
was used. Plot size differed with 11.25 m² in Göttingen
and 9 m² in Einbeck, with a sowing rate of 90 and 110
seeds per m-2, respectively. Fertilization and weed control
were performed in accordance with standard crop man-
agement protocols. Days to flowering (DTF) were
recorded as number of days from sowing till 50% of the
plants in a plot started flowering. At the end of flowering,
fresh biomass yield (FBY kg m-2) was determined by
cutting all plants of a plot 5 cm above the ground with a
harvester and weighing the biomass immediately. Biomass
yield has nearly reached its maximum at this stage, as
demonstrated for Brassica napus by Diepenbrock (2000).
Dry matter content (DMC, %) was determined after drying
a sub-sample of 300 g fresh biomass per plot for 6 days at
60°C in a Memmert ULM 800 drying oven. Dry biomass
yield (DBY g m-2) was calculated on the basis of DMC
and FBY.
Theanalysisofvariance(ANOVA) wasperformedforeach
location as lattice using PLABSTAT software (Utz 2001) and
the model: Yijk=μ+ri+gj+ßk+eijk. Yijkwas defined as the
observation of genotype j in block k and replication i. μ is the
overall mean; riis the effect of replication i (for i=1, 2); gjis
the effect of genotype j (for j=number of genotypes); ßkis
the effect of block k (for k=1, …B); and eijkis the error. A
combined analysis of variance using the adjusted means of
32J Appl Genetics (2012) 53:31–35

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environments was performed for separate groups of
genotypes (parental cultivars, FSwithin, FSbetween, FSwithin
plus FSbetween, and FSmix) based on the model: Yij=μ+li+
gj+lgij+e. Yij is defined as the observation of means of
genotype j in environment l; lidefined as the environmental
effect (l) at location i (for i=1, 2); lgijis the corresponding
Table 2 Mean values of parental
cultivars, full-sibs-within culti-
vars (FSwithin), full-sibs-between
cultivars (FSbetween), and their
mixture (FSmix), as well as the
heterosis of the latter two,
evaluated at two environments
For n, number of genotypes per
trial; DTF, days to flowering;
DMC, dry matter content; FBY,
fresh biomass yield; DBY, dry
biomass yield; SE , standard
error
GroupsnDTF (days)DMC (%)FBY (kg m-2)DBY (g m-2)
CultivarsParent R
Parent L
Parent S
Parental mean
1
1
1
246.76
249.03
246.71
247.50
12.38
11.58
13.92
12.63
3.00
3.07
3.62
3.23
369.05
344.46
399.73
371.08
FSwithin
R×R
L×L
S×S
FSwithinmean
1
1
1
248.75
248.43
246.36
247.85
11.72
11.59
14.06
12.46
3.44
3.41
3.24
3.36
407.96
383.38
457.99
416.45
FSbetween
R×L
R×S
L×S
FSbetweenmean
9
9
9
248.40
247.17
247.00
247.52
12.16
12.69
12.64
12.50
3.59
3.39
3.36
3.45
431.05
433.76
414.55
426.46
FSmix
R×L
R×S
L×S
FSmixmean
1
1
1
248.85
246.37
246.32
247.18
12.79
11.97
12.05
12.27
3.51
3.29
3.49
3.43
433.57
410.82
422.29
422.23
Overall SE 360.600.700.18 28.80
Heterosis
FSbetween(%)
R×L
R×S
L×S
FSbetweenmean
9
9
9
−0.08
−0.16
−0.16
−0.13
4.09
−1.65
−1.57
0.29
4.84
1.49
1.09
2.47
9.33
0.82
−1.21
2.98
Heterosis
FSmix(%)
R×L
R×S
L×S
FSmixmean
1
1
1
0.10
−0.49
−0.44
−0.27
9.17
−7.10
−6.27
−1.40
2.47
−1.52
5.06
2.01
9.67
−5.31
0.43
1.60
Table 1 Mean squares from
combined analysis of variance
of parental cultivars, full-
sibs-within cultivars (FSwithin),
full-sibs-between cultivars
(FSbetween), and their mixture
(FSmix), evaluated in two
environments
For DTF, days to flowering;
DMC, dry matter content; FBY,
fresh biomass yield; DBY, dry
biomass yield. +, *, ** signifi-
cant at P=0.10, P=0.05 and
P=0.01, respectively
Source of variationDf DTF (days) DMC (%)FBY (kg m-2)DBY (g m-2)
Environment (E)
Genotypes (G)
Parental cultivar
FSwithin
FSbetween
Between crosses
Within crosses
FSbetweenvs. FSwithin
FSmix
G×E
Error
1 1163.95**
3.23**
3.50
3.36
3.45**
10.44**
2.87**
0.57
4.15+
0.55+
0.36
180.60**
1.00
2.82
3.89
0.78
1.56
0.72
0.01
0.40
0.71
0.48
2.97**
0.09
0.22
0.02
0.09
0.28*
0.07
0.04
0.03
0.07**
0.03
49783.79**
1548.45
1533.57
2891.70
1082.43
1946.39
1010.44
541.10
259.01
1453.67*
829.36
35
2
2
26
2
24
1
2
35
50
J Appl Genetics (2012) 53:31–35 33

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interaction effect; and e is the pooled error from lattice
experimental analyses at the two locations. The environment
was considered as fixed. In the ANOVA, FSbetweeneffects
were partitioned into ‘between crosses’ with (3−1) degreesof
freedom and ‘within crosses’ with 3 × (9−1) degrees of
freedom. The FSbetweenvs. FSwithineffects (Table 1) were
calculated based on the sum of squares (SS): SSFSbetween vs
FSwithin=SSFSwithin plus FSbetween−SSFSwithin−SSFSbetween.
Heterosis was estimated as mid-parent heterosis and
FSwithinwas used as parental value.
For all biomass parameters DMC, FBY and DBY, the
combined ANOVA (Table 1) revealed the environment as
largest effect and significant genotype × environment-
interactions for beginning of flowering (days to flowering;
DTF), DBY and FBY. For the interpretation of the latter, it
has to be considered, that the experimental basis is rather
small with only two environments.
The parental cultivars Rex, Largo, and Steinacher
varied in fresh and dry biomass parameters (Table 2).
The eldest cultivar Steinacher showed the highest DMC
with 13.92% as parent and 14.06% for FSwithin S×S,
respectively, whereas the more recently released cultivar
Largo revealed the lowest DMC and DBY. Significant
differences were detected in yield parameter FBY and in
flowering time (Table 1) between the three FSbetween
crossing combinations.
The mean performance of FSwithin, which was devel-
oped by randomly crossing individual plants in a popula-
tion, is expected to be the same as the parental cultivar
mean (Falconer and Mackay 1996). FSwithin produced
higher biomass yields than the parental cultivars. This may
have been the result of unconscious positive selection for
better plants for the production of FSwithin. Moreover, the
seeds of parental lines were not produced in the same
environment as FSbetween, FSwithinand FSmix. For these
reasons, FSwithinwas used instead of the parental cultivars
for the estimation of heterosis; otherwise, heterosis might
have been overestimated.
The mean values of biomass yield parameters (FBY and
DBY) increased in general as expected due to heterosis:
FSwithin< FSmix< FSbetween.
A small andpositivemid-parentheterosis wasobservedfor
the meanvalues ofthe yield traitsFBYand DBY for FSbetween
(Table 2). The avarage mean heterosis effects of FSbetween
were small and the largest effect was observed in FSbetween
crossing combination R×L with 4.84% for fresh and 9.33%
for dry biomass yield (Table 2). Heterosis estimations of
individual FSbetween revealed a maximum of 14.58% for
FBY and 19.65% for DBY and a minimum of −7.46%
and −12.68%, respectively.
The mean mid-parent heterosis effects of FSbetween
exceeded FSmix in both yield parameters fresh and dry
matter yield. The maximum heterosis for fresh biomass
yield was observed in FSmix crossing combination L×S
with 5.06% and for dry matter yield in FSmix crossing
combination R×L with 9.67% (Table 2). Minimum heter-
osis was negative with −1.52 for fresh and −5.31 for dry
matter yield. The small negative heterosis for flowering
time (DTF) showed that FSbetweenflowered slightly earlier
than the corresponding parental FSwithin.
The low amount of heterosis in crosses between the
three cultivars Rex, Steinacher and Largo, supports
earlier results with molecular markers, revealing that
most of the genetic diversity is within cultivars (83%)
compared to only 17% between cultivars (Ofori et al.
2008). In this experiment, only three cultivars were used,
but they represent nearly 50 years of European winter B.
rapa breeding. They differ completely in seed quality,
which is of no importance for biomass use, and show the
maximum diversity which can be expected in the
European genepool. Other studies with six (Zhao et al.
2009) or ten (Zhao and Becker 1998) cultivars corroborate
the low diversity in European winter cultivars and suggest
to broaden the diversity by utilizing Chinese genetic
resources.
Self-incompatibility (SI) supports cross pollination by
rejection of self-pollen. In B. rapa, a multiallelic gene
complex at the S-locus controls the SI recognition and a
number of different S-alleles differing in intensity have
been described (Sakamoto and Nishio 2001). In this study,
FSbetweencrosses were produced without emasculation by
isolation of two plants, assuming that the plants were self-
incompatible. Hybridity was confirmed by using seed
erucic acid content as a marker. The selection for different
S-alleles in parental cultivars could be a basis for future
inter-population or inter-varietal crosses, which would
increase the level of hybridity and reduce the amount of
within cultivar pollinations.
It could be assumed that heterosis in crosses between
cultivars (FSbetweenand FSmix) was low because of the low
genetic variation between and the high genetic variation
within the cultivars. Therefore, it is not very promising to
develop populations of synthetic cultivars by combining
lines from different European winter cultivars. Increasing
the amount of heterosis by including less closely related
cultivars or genotypes of the B. rapa genepool, such as
Chinese genotypes (Zhao and Becker 1998; Zhao et al.
2009), could be an option.
Acknowledgements
grant of FNR (Agency for Renewable Resources) to KWS Saat AG,
Einbeck. We thank Dr. Andreas Gertz for fruitful discussions.
The project was financially supported by a
Open Access
Creative Commons Attribution Noncommercial License which per-
mits any noncommercial use, distribution, and reproduction in any
medium, provided the original author(s) and source are credited.
This article is distributed under the terms of the
34J Appl Genetics (2012) 53:31–35

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