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SID 5



Research Project Final Report
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SID 5 (Rev. 3/06)
Project identification
NF0532
Brassica alboglabra -developing a new oil crop for the
UK
Contractor
organisation(s)
Warwick HRI
University of Warwick
Wellesbourne
Warwick
CV35 9EF
54. Total Defra project costs
(agreed fixed price)
5. Project:
Page 1 of 11
£
44,893
start date ................
01 January 2007
end date .................
31 March 2007
6. It is Defra’s intention to publish this form.
Please confirm your agreement to do so. ................................................................................... YES
NO
(a) When preparing SID 5s contractors should bear in mind that Defra intends that they be made public. They
should be written in a clear and concise manner and represent a full account of the research project
which someone not closely associated with the project can follow.
Defra recognises that in a small minority of cases there may be information, such as intellectual property
or commercially confidential data, used in or generated by the research project, which should not be
disclosed. In these cases, such information should be detailed in a separate annex (not to be published)
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Executive Summary
7.
The executive summary must not exceed 2 sides in total of A4 and should be understandable to the
intelligent non-scientist. It should cover the main objectives, methods and findings of the research, together
with any other significant events and options for new work.
The work carried out in this three month project had the aim to demonstrate the feasibility of developing
Brassica alboglabra which is a white flowered form of Brassica oleracea as a new industrial oil crop for the
UK.
The project had three objectives:
1. To demonstrate the existence of natural variation within B.oleracea and related wild
species for seed oil fatty acid profile and seed oil content.
2. To identify allelic variation for genes associated with fatty acid biosynthesis.
3. To develop an ideotype for Brassica alboglabra as a new UK oil crop and identifying
the gaps in knowledge required to produce that ideotype.
The results described below clearly indicate that it would be feasible to develop a Brassica oleracea oil
crop for the UK. We have demonstrated the existence within the C genome genepool (B. oleracea and
related wild species) greater natural variation for seed oil fatty acid profile and seed oil content than was
found in the Ac (B. napus) genepool. We have also demonstrated allelic variation for genes known to be
associated with fatty acid biosynthesis in the C genome genepool. This provides the underpinning basis
for developing an oil crop in which cultivars are bred to produce oils tailored for specific industrial end
uses. The ‘plasticity’ of the C genome as demonstrated by the many and varied crop morphologies within
the species and also the variation in morphology between the wild relatives provides the genetic basis for
redesigning a crop ideotype to provide a new oil crop which meets stakeholder needs in terms of
agronomy, yield and reduced (potentially beneficial) environmental impact. However, a more extensive
follow up project would be required to underpin. This would exploit the range of experimental genetic and
genomic resources in C genome brassica available at Warwick HRI in a pre-breeding project to deliver
plant lines with combinations of alleles to give optimised fatty acid profiles for particular end uses and
optimised sustainable crop production i.e reduced and potentially beneficial environmental impact. These
could be subsequently exploited in a programme to breed a novel (B oleracea) oil crop for the UK or
delivered to the current UK oil seed breeding community by introgressing them via interspecific crosses
and embryo rescue into a common ‘crop portal’ which would be an experimental line(s) derived from a
well adapted cultivar(s) to be identified by the OSR group of the British Society of Plant Breeders.
SID 5 (Rev. 3/06)
Page 2 of 11
Objective 1: To demonstrate the existence of natural variation within B. oleracea and related wild
species for seed oil fatty acid profile and seed oil content. Natural variation for seed oil fatty acid
profile and content was demonstrated by analysing the seed oil of 10 seeds of each of the gene bank
accessions used to make the diversity sets for cultivated forms of Brassica, oleracea and 14 related wild
species (oleracea; alboglabra; atlantica, balearica; bourgaei; cretica; hilaronis; incana; insularis;
macrocarpa; montana; robertiana; rupestris; villosa) which are being produced in HH3723 and the
diversity set for B. napus which is being produce din the OREGIN project. Significant variation was found
between accessions both for the range and amount of different fatty acids. The greatest observed
variation was between the accessions in the wild C genome diversity set with the least variation being
found in the B.napus set. This can be explained due to the effect of strong selection pressure which has
been exerted by breeders on the modern B. napus cultivars for high oleic acid and low erucic acid to meet
market needs; whereas the wild C genome species will not have been subjected to such strong
unidirectional selection. The domesticated C genome genepool (B.oleracea diversity set) displayed less
variation than the wild species suggesting that some variation may have been lost during domestication.
Objective 2: To identify allelic variation for genes associated with fatty acid biosynthesis:
A number of genes involved in the biosynthesis pathway of fatty acids have been identified in the model
dicotyledon plant and member of the Brassicaceae, Arabidopsis thaliana. Three of these genes which are
important in fatty acid synthesis: fatty acid elongase (FAE) and two fatty acid desaturases (FAD2 and
FAD3) were sequenced in 188 accessions of the B. oleracea diversity set, and 96 accessions from both
the wild C genome species and the B.napus diversity sets. The sequences were analysed for changes
which may affect protein structure and hence enzyme function i.e. have an effect on fatty acid profile and
content. A significant number of different alleles were identified for each gene. The wild C genome species
DFFS and B oleracea DFFS sets showed a similar range of allelic variants with the B.napus DFFS set
exhibiting the least diversity.
Objective 3: To develop an ideotype for Brassica alboglabra as a new UK oil crop and identifying
the gaps in knowledge required to produce that ideotype. There is a large diversity of crop
morphology within B. oleracea; there is therefore potential to manipulate crop architecture in order to
optimise the crop. In addition there are the tools and resources available in B. oleracea to exploit genetic
variation for important input traits such as nitrogen, phosphorous and water use efficiency as well as pest
and disease resistance. It is therefore possible to ‘design’ and breed to an ideotype to maximise the
sustainability of the crop as a renewable feedstock. The current information relating to vegetable forms of
B. oleracea was reviewed together with information from OSR studies during a ‘brain storming’ session to
identify where it is applicable to the development of an oil form of the crop. This clearly demonstrated the
potential of utilising the greater ‘plasticity’ of the C genome (as opposed to the napus genome) to breed a
crop with many desirable traits. Although the C genome contains many gene duplications and triplications
the effort required to understand and manipulate plant morphology will be easier in this ‘diploid’ species
than in an allotetraploid such as B napus where the degree of gene duplication is doubled. Aspects of the
crop which will were examined included:












Spring sown v autumn sown crops (vernalisation requirement)
Canopy structure/branching
Flowering time
Harvest index
Height
Root architecture
Energy balance/efficiency (input v output)
Yield
Pod shatter
Pest and disease resistance
Resource use efficiency
Breeding system (hybrid v inbred line)
These were considered from a viewpoint of adaptation to climate change, carbon balance, economics,
land use, environmental footprint and public perception.
SID 5 (Rev. 3/06)
Page 3 of 11
Project Report to Defra
8.
As a guide this report should be no longer than 20 sides of A4. This report is to provide Defra with
details of the outputs of the research project for internal purposes; to meet the terms of the contract; and
to allow Defra to publish details of the outputs to meet Environmental Information Regulation or
Freedom of Information obligations. This short report to Defra does not preclude contractors from also
seeking to publish a full, formal scientific report/paper in an appropriate scientific or other
journal/publication. Indeed, Defra actively encourages such publications as part of the contract terms.
The report to Defra should include:
 the scientific objectives as set out in the contract;
 the extent to which the objectives set out in the contract have been met;
 details of methods used and the results obtained, including statistical analysis (if appropriate);
 a discussion of the results and their reliability;
 the main implications of the findings;
 possible future work; and
 any action resulting from the research (e.g. IP, Knowledge Transfer).
The work carried out in this three month project had the aim to demonstrate the feasibility of developing Brassica
alboglabra which is a white flowered form of Brassica oleracea as a new industrial oil crop for the UK.
The project had three objectives:
4. To demonstrate the existence of natural variation within B.oleracea and related wild species
for seed oil fatty acid profile and seed oil content.
5. To identify allelic variation for genes associated with fatty acid biosynthesis.
6. To develop an ideotype for Brassica alboglabra as a new UK oil crop and identifying the gaps
in knowledge required to produce that ideotype.
Objective 1: To demonstrate the existence of natural variation within B. oleracea and related
wild species for seed oil fatty acid profile and seed oil content.
Diversity fixed foundation sets (DFFS) are being developed under HH3723 for cultivated Brassica
oleracea and 14 wild related wild ‘C genome’ species (oleracea; alboglabra; atlantica, balearica;
bourgaei; cretica; hilaronis; incana; insularis; macrocarpa; montana; robertiana; rupestris; villosa).
These are defined as “Informative sets of genetically fixed (true breeding) lines representing a
structured
sampling
of
diversity
within
the
relevant
genepool”
(http://www.brassica.info/diversity/diversity_sets.ht). Each line within the DFFS is being derived from a
single accession from the Genetic Resources Unit (GRU) international brassica collection at Warwick
HRI and these ‘founder’ accessions were used as the experimental material for the survey of fatty acid
profile in seed oil and oil content.
Ten seeds of each accession were obtained either from the GRU (for B. oleracea accessions) or from
S1 seed lots produced by self-polinating the C genome wild species founder plants in HH3723. All seed
were equilibrated at 150C and 15% relative humidity prior to sampling. Individual seeds were weighed
in 2 mL glass analysis vials with sealable lids. At the sampling stage seed were handled with nitryl
gloves to reduce contamination. Lipid fatty acids were converted to their methyl esters (FAMEs) using
the direct transmethylation method and GC equipment as described by (Larson and Graham, 2001),
with some modifications. To minimize seed to seed variation, 10 seeds were analysed for each
accession and processed through the transmethylation procedure. Heptadecanoic acid (100 µg) was
added as an internal standard before processing. Cooled transmethylated samples were transferred to
microfuge tubes and extracted three times with 200 µL hexane, with 1 min centrifugation at 14000xg
between each extraction step to clarify the partitioned layers. The hexane fractions were pooled, dried
in vacuo for 10 min and reconstituted in 1 mL fresh hexane. This procedure demonstrated comparable
quantitative extraction for 1-5 seeds, with no losses for FAMEs with acyl chains >C10 (data not shown).
A 2 µL aliquot was injected for FAME analysis. Fatty acids were identified and quantified by
comparison to a 37 FAME mix (Supelco), using Chromquest 2.53 software (Thermo). The absolute and
relative amounts of each fatty acid were calculated.
SID 5 (Rev. 3/06)
Page 4 of 11
The analysis of fatty acid profile demonstrated there was significant variation between accessions for
the range and amount of different fatty acids. Figure 1 shows examples of the
22:1n
9
18:1n9
c
20:1n
9
18:3n
3
18:2n6
c
B. oleracea gemmifera
(Brussels Sprout)
18:1n9
c
18:1n9
c
22:1n
9
18:2n6
c
22:1n
9
18:3n
3
18:3n
3
B. oleracea italica
(Inbred Broccoli)
18:2n6c
B. oleracea italica
(Doubled haploid Broccoli)
Fig 1: % FA mol
variation
between
Brassica
oleracea
accessions
variation seen in fatty acid profile between accessions of cultivated B. oleracea and figure 2 shows the
variation seen in the amount of an individual fatty acid (18:2n6 - Omega 6 ) as a percentage of the total
seed fatty acid content.
35
30
Fig 2. Amount of
18:2n6 (Omega 6)
as a percentage of
the total seed fatty
acid content in 192
accessions of B
oleracea
%mol
25
20
15
10
5
0
1
11 21 31 41
51 61 71 81 91 101 111 121 131 141 151 161 171 181 191
Plant line
Figure 3 further demonstrates the natural variation found within three genepools: the wild C genome
genepool (fig 3a) the cultivated C genome (B. oleracea) genepool (Fig 3b) and the cultivated AC (B.
napus) genepool for an individual fatty acid; this time for Omega 3 (18:3n3) fatty acid.
SID 5 (Rev. 3/06)
Page 5 of 11
Fig 3. Amount of 18:3n3 (Omega 3) as a percentage of the total seed fatty acid content in
a) Brassica spp “C” genome DFFS (Wild species) set 1
35.00
30.00
%mol
25.00
20.00
15.00
10.00
5.00
C
07
0
C 01
07
0
C 18
07
0
C 11
07
0
C 13
07
0
C 21
07
0
C 28
07
0
C 22
07
0
C 29
07
0
C 31
07
0
C 39
07
0
C 38
04
0
C 31
07
0
C 51
07
0
C 61
04
0
C 57
07
C 062
07
07
C 9A
07
0
C 59
07
1
C 21
07
0
C 55
07
0
C 63
07
0
C 56
07
0
C 89
04
0
C 90
04
0
C 93
04
0
C 87
04
08
4
0.00
b) Brassica oleracea DFFS set 1
30.00
%mol 18:3n3
25.00
20.00
15.00
10.00
5.00
ge
ra
m
m
go ifer
a
ng
ylo
de
s
ita
lic
a
ita
lic
a
ita
lic
a
ita
lic
a
ol
ife
ra
tro
nc
hu
da
ta
ge
m
m
ife
ta
pi
ta
ca
ta
pi
ta
ca
p
pi
ta
ca
p
sp
C
tis
sp
C
tis
bo
t ry
tis
bo
t ry
tis
bo
t ry
tis
bo
t ry
bo
t ry
a
al
a
br
al
bo
gl
a
al
a
ep
h
ac
ep
h
ac
ac
ep
h
al
a
0.00
c) Brassica napus DFFS set 1
25.00
20.00
15.00
10.00
5.00
A
ge
n
DH om
e
Sw
ed
sp
r
rin a p e
g
e
fo
k
ra ale
g
Sp e ra
rin pe
g
Sp O
rin SR
g
Sp OS
rin
R
g
Sp O
rin SR
g
Sp OS
rin
R
g
Sp OS
rin
R
g
Sp O
rin SR
g
O
SR
sw
ed
e
sw
ed
e
sw
ed
sw
e
s
e
wi de wed
nt
l
er an e
fo dra
dd
ce
e
W r ra
in
pe
te
rO
W
S
in
te R
W rO
S
in
te R
rO
W
S
in
te R
W rO
S
in
te R
rO
W
S
in
te R
W rO
S
in
te R
rO
W
S
in
te R
rO
W
S
in
te R
W rO
S
in
te R
rO
W
S
in
te R
W rO
S
in
te R
rO
SR
0.00
This fatty acid was chosen because selection was not expected to have had much influence on this
fatty acid, however, evidence for a significant effect of selection was found. The greatest observed
variation was between the accessions in the wild C genome DFFS set with the least variation being
found in the B.napus DFFS set. This can be explained due to the effect of strong selection pressure
which has been exerted by breeders on the modern B. napus cultivars for high Oleic acid (18:1n9) and
SID 5 (Rev. 3/06)
Page 6 of 11
low Erucic acid (22:1n9) to meet market needs which presumably has had an indirect effect on the
content of 18:3n3; whereas the wild C genome species will not have been subjected to such strong
unidirectional selection. It is interesting to note that the domesticated section of the C genome
(B.oleracea) displays less variation than the wild species suggesting that some variation may have
been lost during domestication.
It is clear from the data that we have generated in this preliminary study that there is significant
natural variation for both fatty acid profile and amount of individual fatty acids within the seed
oil of B. oleracea and its close wild relatives.
Objective 2: To identify allelic variation for genes associated with fatty acid biosynthesis
A number of genes involved in the biosynthesis pathway of fatty acids have been identified in the
model dicotyledon plant and member of the Brassicaceae, Arabidopsis thaliana. These include the fatty
acid desaturase genes FAD3 (AT2g29980) and FAD2 (AT3g12120) (Okuley et al., 1994), fatty acid
elongases FAE1 (At4g34520) and FAE homologue (At3g52160), palmitoyl-ACP thioesterase FatB
(At1g08510) and the acyl carrier protein ACP1 (At3g11430) (Bonaventure and Ohlrogge, 2002).
Closely related orthologues of these genes have been isolated from Brassica species. The B. napus
genes FAE1.1 and FAE1.2 have been shown to co-segregate with two loci which govern erucic acid
production (Barret et al., 1998; Fourmann et al., 1998), with FAE1 genes also being isolated from B.
oleracea and B. rapa (Das et al., 2002) as well as B. juncea (Gupta et al., 2004). Between four and six
copies of FAD2 have been reported for B. napus (Scheffler et al., 1997) and representative orthologous
genes encoding for both FAD2 and FAD3 have been cloned (Arondel et al., 1992; Okuley et al., 1994)
In order to amplify locus specific regions of the paralogues of three significant genes involved in fatty
acid synthesis (FAE, FAD2 and FAD3) locus-specific primers were designed to an approximately 1000
bp segment of the coding region sequence. These were amplified with a proof-reading PCR
polymerase to produce regions of the FAE and FAD genes from a structured set of 188 accessions of
the B. oleracea DFFS, and 96 accesions from the C genome DFFS and the B.napus DFFS. The
amplification products were sequenced from both strands and the sequences analysed for single
nucleotide polymorphisms (SNP’s), synonymous and non-synonymous changes and for the presence
of indels which may affect protein function. A significant number of different alleles were identified for
each gene. The wild C genome spp DFFS and B oleracea DFFS sets showed a similar range of allelic
variants with the B.napus DFFS set exhibiting the least diversity.
a) Screening for allelic variants of FAE1-2
A significant number of allelic variants were detected in a Fae_1-2 C genome locus
Shown here is variation in sequence found between AA132-148
TTCGGGTCTAGGCGATGAAACCCACGGGCCCGAGGGGCTGCTTCATGTCC
____A_____________________T______________________________.G____
This region showed two synonymous and one non synonymous allelic differences
The following illustrates the effect of the above allelic substitutions in the FAE 1-2 gene on the amino
composition of part of the protein
134 GLGDETHGPEGLLQVPPRKTFAAAREETE 162
134 GLGDETHGPEGLLHVPPRKTFAGAREETE 162
Non synonymous changes were found at amino acid positions 147, 156,181, 214, 234 in many
accessions. Synonymous changes were also often shared between multiple accessions. Additional
allelic variants, both synonymous and non-synonymous, were also found which were unique to
individual lines. In contrast when the same primers are used to amplify this region of FAE1-2 from the
B.napus DFFS only one accession was found to contain an allelic variant. This illustrates the
effectiveness of the Brassica oleracea DFFS and the wild C genome DFFS as sources of potentially
useful allelic variation which could be introgressed into B.napus.
SID 5 (Rev. 3/06)
Page 7 of 11
b) Screening for allelic variants of FAD-2
A similar degree of allelic variation was detected within a FAD-2 locus
CTCTTCCGTTACGCCGCCGCGCAGGGAGTGGCCTCGATGGTCTGCTTTTACGGAGTC
____A________________________A_____________________________C________
Within the same region 28 accesions had alternative base pair substitutions at AA 247. Substitution sat
positions 255 and 261 appeared correlated but had no effect on the AA. 32 out of 96 lines showed nonsynonymous change at AA241.
FAD2 AA239-261
GLFRYAAAQGVASMVCFYGVPLLIVN
GLYRYAAAQGVASMVCFYGVPLLIVN
Outside this region of this FAD2 locus a further 5 SNPs were detected which appeared to be non
synonymous.
c) Screening for allelic variants of FAD-3
In contrast to FAE1-2 and FAD2 the PCR results with a FAD3 locus suggested that there was no nonsynonymous changes detected. Further work will be required to verify this result.
Allelic variants identified by sequence analysis were matched against fatty acid profiles to determine if
there were any correlations between sequence variation and long chain fatty acid synthesis. It was
difficult to detect any direct relationships due to the number of allelic variants found with no obvious link
with the non–synonomous changes in the FAE1-2. However, the results of the preliminary allelic survey
for three loci known to affect fatty acid biosynthesis clearly demonstrated allelic variation for genes
associated with fatty acid biosynthesis.
Objective 3: To develop an ideotype for Brassica alboglabra as a new UK oil crop and
identifying the gaps in knowledge required to produce that ideotype.
There is a large diversity of crop morphology within B. oleracea; there is therefore potential to
manipulate crop architecture in order to optimise the crop. In addition there are the tools and resources
available in B. oleracea to exploit genetic variation for important input traits such as nitrogen,
phosphorous and water use efficiency as well as pest and disease resistance. It is therefore possible
to ‘design’ and breed to an ideotype to maximise the sustainability of the crop as a renewable
feedstock.
The current information relating to vegetable forms of B. oleracea was reviewed this together with
information from OSR studies to identify where it is applicable to the development of an oil form of the
crop. We also identified where additional information is required. Aspects of the crop which will need to
be examined include:












Spring sown v autumn sown crops (vernalisation requirement)
Canopy structure/branching
Flowering time
Harvest index
Height
Root architecture
Energy balance/efficiency (input v output)
Yield
Pod shatter
Pest and disease resistance
Resource use efficiency
Breeding system (hybrid v inbred line)
SID 5 (Rev. 3/06)
Page 8 of 11
These were considered from a viewpoint of adaptation to climate change, carbon balance, economics,
land use, environmental footprint and public perception.
An initial brain storming session on the ‘ideal plant’ was carried out; the results of this are given in the
appendix. This was the only activity aimed at this objective within the resources of the project. It will
need to be taken further and to be informed by results from other projects currently being carried out on
OSR and B. oleracea – e.g nitrogen use efficiency LINK project, Phosphorous use efficiency project in
OSR and B. oleracea; Novorb LINK project etc. However, it clearly demonstrates the potential of
utilising the greater ‘plasticity’ of the C genome (as opposed to the napus genome) to breed a
crop with many desirable traits suited to a more sustainable low input production system.
Although the C genome contains many gene duplications and triplications the breeding effort required
to manipulate plant morphology will be easier in a ‘diploid’ species than in an allotetraploid such as B
napus where the degree of gene duplication is doubled.
Conclusion:
This three month preliminary project had the overall aim to demonstrate the feasibility of developing
Brassica alboglabra which is a white flowered form of Brassica oleracea as a new industrial oil crop for
the UK. The results obtained clearly indicate that it would be feasible to develop a Brassica oleracea oil
crop for the UK. We have demonstrated the existence within the C genome genepool (B. oleracea and
related wild species) greater natural variation for seed oil fatty acid profile and seed oil content than
was found in the Ac (B. napus) genepool. We have also demonstrated allelic variation for genes known
to be associated with fatty acid biosynthesis in the C genome genepool. This provides the
underpinning basis for developing an oil crop in which cultivars are bred to produce oils tailored for
specific industrial end uses. The ‘plasticity’ of the C genome as demonstrated by the many and varied
crop morphologies within the species and also the variation in morphology between the wild relatives
provides the genetic basis for redesigning a crop ideotype to provide a new oil crop which meets
stakeholder needs in terms of agronomy, yield and reduced (potentially beneficial) environmental
impact.
The project has therefore clearly demonstrated the feasibility and potential of developing a form
of Brassica oleracea as a new industrial oil crop for the UK. It has also clearly demonstrated
that the C genome genepool is a source of potentially useful genetic variation for improving the
UK OSR crop. However, there is a need for a more extensive future project to provide the
underpinning knowledge to undertake either of these routes.
Future work:
A future project would exploit the range of experimental genetic and genomic resources in C genome
brassica available at Warwick HRI. We are currently developing the Diversity Sets (DS) for B. oleracea
and wild C genome Brassica spp related to B. oleracea. These unique structured samples of the
genetic resource collections would be used to extend the current study of the natural genetic variation
within the C genepool at both the phenotypic level and the level of allelic variation for ‘key’ genes in oil
biosynthesis.
Warwick HRI (W HRI) also maintains a number of defined Brassica mapping populations of double
haploid (DH) and substitution lines under both the OREGIN (Defra) and AdVaB (BBSRC Crop Sci)
projects. (QTL analysis using two of our B.oleracea mapping populations have already identified 22
QTL associated with metabolic step changes in FA synthesis). A wide selection of brassica genomic
libraries (EST, Genomic and BACs) are available at W HRI and considerable effort has been put into
adding value to these though physical mapping (BBSRC, IGF Project), hybridisations of macroarrays
and BAC end sequencing. W HRI is also directly involved in the multinational Brassica genome
sequencing programme (MBGP) funded through BBSRC. We are therefore in a position to move
from the identification of QTL to the identification of the genes responsible for the traits of
interest. Carrying out these studies in B oleracea avoids the problems associated with the additional
level of gene duplication in tetraploid B. napus. We will use this knowledge in conjunction with our
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knowledge of the pathways involved in FA synthesis and catabolisim, to design a pre-breeding program
to combine alleles to provide a FA profiles matching those identified by chemists and materials
engineers as optimal for different end uses. In addition we will also exploit our existing knowledge of
the genetics of seed size and oil content and other components of yield to maximize the energy
embodied in the oil.
The C genome is also a strategic source of beneficial alleles for traits with an environmental
benefit. We have already demonstrated the value of the B. oleracea diversity set in studies to assess
variation at the phenotypic level for traits such as phosphorous use (PUE) and water use efficiency
(WUE). We hypothesise that the C genome wild species DS is a potentially rich source of beneficial
alleles for ‘traits for sustainable production’ as these species have evolved under natural low input
conditions.
We will survey for allelic variation at the sequence level in key genes associated with traits for
sustainability with the aim of identifying allelic variants associated with phenotypic variation in order to
provide beneficial alleles for breeding for increased sustainability of production. This approach will
increase the efficiency of identifying potential beneficial alleles since only those accessions possessing
allelic variants need to be phenotyped. We have initiated a demonstration of this approach within the
OREGIN project with an allelic survey of a subset of the B. napus DS for one of the cytoplasmic
isoforms and one of the plastidic isoforms of glutamine synthetase. However, this study has shown the
difficulty of attempting to identify allelic variation in an allotetraploid. We will therefore use a strategy of
initial gene identification and function studies in the genetically less complex diploid species rather than
the allotetraploid. All of the species are compatible with B. oleracea and so novel allelic variants
can be introgressed into the domesticated genepool either directly to vegetable brassicas or via
inter-specific hybridisation and embryo rescue to OSR.
We will deliver the outputs of this ‘pre breeding’ programme to the oil seed breeding community
by introgressing them into a common ‘crop portal’. This would be a DH line(s) derived from a well
adapted cultivar(s) to be identified by the OSR group of the British Society of Plant Breeders.
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References to published material
9.
This section should be used to record links (hypertext links where possible) or references to other
published material generated by, or relating to this project.
Arondel V, Lemieux B, Hwang I, Gibson S, Goodman HM, Somerville CR (1992) Map-Based Cloning
of a Gene Controlling Omega-3-Fatty-Acid Desaturation in Arabidopsis. Science 258: 1353-1355
Barret P, Delourme R, Renard M, Domergue F, Lessire R, Delseny M, Roscoe TJ (1998) A rapeseed
FAE1 gene is linked to the E1 locus associated with variation in the content of erucic acid.
Theoretical and Applied Genetics 96: 177-186
Bonaventure G, Ohlrogge JB (2002) Differential regulation of mRNA levels of acyl carrier protein
isoforms in Arabidopsis. Plant Physiology 128: 223-235
Das S, Roscoe TJ, Delseny M, Srivastava PS, Lakshmikumaran M (2002) Cloning and molecular
characterization of the Fatty Acid Elongase 1 (FAE 1) gene from high and low erucic acid lines of
Brassica campestris and Brassica oleracea. Plant Science 162: 245-250
Fourmann M, Barret P, Renard M, Pelletier G, Delourme R, Brunel D (1998) The two genes
homologous to Arabidopsis FAE1 co-segregate with the two loci governing erucic acid content in
Brassica napus. Theoretical and Applied Genetics 96: 852-858
Gupta V, Mukhopadhyay A, Arumugam N, Sodhi YS, Pental D, Pradhan AK (2004) Molecular tagging
of erucic acid trait in oilseed mustard (Brassica juncea) by QTL mapping and single nucleotide
polymorphisms in FAE1 gene. Theor Appl Genet 108: 743-749
Larson TR, Graham IA (2001) Technical Advance: a novel technique for the sensitive quantification of
acyl CoA esters from plant tissues. Plant J 25: 115-125
Okuley J, Lightner J, Feldmann K, Yadav N, Lark E, Browse J (1994) Arabidopsis FAD2 gene encodes
the enzyme that is essential for polyunsaturated lipid synthesis. Plant Cell 6: 147-158
Scheffler JA, Sharpe AG, Schmidt H, Sperling P, Parkin IAP, Lühs W, Lydiate DJ, Heinz E (1997)
Desaturase multigene families of <SMALL>Brassica napus</SMALL> arose through genome
duplication. TAG Theoretical and Applied Genetics 94: 583
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