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SID 5



Research Project Final Report
Note
In line with the Freedom of Information
Act 2000, Defra aims to place the results
of its completed research projects in the
public domain wherever possible. The
SID 5 (Research Project Final Report) is
designed to capture the information on
the results and outputs of Defra-funded
research in a format that is easily
publishable through the Defra website. A
SID 5 must be completed for all projects.
1.
Defra Project code
2.
Project title
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boxes may be expanded or reduced, as
appropriate.
3.
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SID 5 (Rev. 3/06)
Project identification
IF0168
Improved resolution of QTL associated with Water and P
use efficiency
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 6
£
49,810
start date ................
01 October 2008
end date .................
31 March 2009
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 In these cases, such information should be detailed in a separate annex (not to be published)
so that the SID 5 can be placed in the public domain. Where it is impossible to complete the Final Report
without including references to any sensitive or confidential data, the information should be included and
section (b) completed. NB: only in exceptional circumstances will Defra expect contractors to give a "No"
answer.
In all cases, reasons for withholding information must be fully in line with exemptions under the
Environmental Information Regulations or the Freedom of Information Act 2000.
(b) If you have answered NO, please explain why the Final report should not be released into public domain
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.
Water is the most important factor limiting crop production. Crop production in the UK is either rain-fed, as
in the case of the majority of arable crops, or relies on supplementary irrigation. The total amount of water
abstracted for irrigation of field crops is around 130 million cubic meters (Mm 3), with protected edible crops
and ornamentals using approximately another 60 Mm 3 for irrigation. Water availability during the UK
growing season is predicted to decline and may make some non-irrigated crop production economically
unsustainable. In addition the excessive use of soil water by non-irrigated crops reduces the amount of
rain water returned to surface or ground water reserves. Irrigated crops will also require a greater input of
supplementary irrigation to maintain production because of both reductions in rain-fall and increases in
evapotranspiration as average temperatures increase. Consequently, there is a need to minimise the
amount of water required to grow crops, and to this end it will be important to have crop varieties that can
use the available water more efficiently.
In addition to water, most crops require high inputs of inorganic fertilisers to maintain yield and quality.
These fertilisers are energy intensive to produce and can (if not correctly managed) pollute adjacent
environments leading to a loss of biodiversity. Capture of nutrients from the soil is a key crop trait that
impacts on the quantity of fertilizer that needs to be applied to soil and the entry of nutrients into water
courses. Development of crop varieties that can maintain yields with lower fertiliser inputs and that recover
more applied nutrients are key to the development of low input agricultural systems.
Previous work (HH3501, WQ0119, HH3608TX, LK0979, IF0125, HH3608TX, WU0116), has identified
QTL associated with water-use efficiency in the Brassica oleracea genome and validated this in the
substitution line AGSL118, which differs from the recurrent parental line (A12DHd), by substitutions of
genomic DNA from the other parent (GD33) in linkage group, C1, C6 and C7. Several QTL have also been
identified for various measures of phosphorus use efficiency (HH3501) on linkage groups C3 and C7 of B.
oleracea in a number of substitution lines.
In order to define the position of these QTL more precisely, back-crosses to the A12 parent have been
performed and stocks of F1 or F2 seed produced, depending on the lines. For example, previously
AGSL118 was crossed to A12, and the resulting line (BC 1F1) was grown and allowed to self-pollinate to
give the BC1F2 seed stock. The aim of this project was to identify recombinants in the BC 1F2 population of
the cross A12 x AGSL118 using molecular markers. A panel of homozygous recombinant lines would then
be assessed for phenotype to establish a single small mapping interval for each QTL.
SID 5 (Rev. 3/06)
Page 2 of 6
This current project had two objectives to facilitate the continued back-crossing of these lines to fine map
QTL associated with water and phosphorus use efficiency. In Objective 1, a total of 421 BC1F2 plants from
the introgression line AGSL118 were sown in the glasshouse and sampled to obtain DNA. PCR
amplifications were made on these DNA samples using the primer pairs for markers identified as close as
possible to the proximal and distal ends of the introgressed regions in line AGSL118. In initial screens, two
classes, either single or double introgressions, of plant genotype were selected and retained for self
pollination and seed production. Of these approximately 70 of these plants, representing all genotypes,
have been grown to flowering and BC1F3 seed will be collected within two months. In Objective 2, a total of
17 potential markers spanning the map interval of the introgressed regions for AGSL118 were screened
on GD33 and A12DHd parental and BC1F1 (heterozygous AGSL118) DNA. Three of these markers were
associated with linkage group 1, five with linkage group 6 and nine with linkage group 7. Four markers
shown to be polymorphic between the two parents and within the introgressed GD33 regions of AGSL118
were selected for screening our BC1F2 plants.
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).
Background
Water is the most important factor limiting crop production. Crop production in the UK is either rain-fed, as in the
case of the majority of arable crops, or relies on supplementary irrigation. The total amount of water abstracted for
irrigation of field crops is around 130 million cubic meters (Mm 3), with protected edible crops and ornamentals
using approximately another 60 Mm 3 for irrigation. Water availability during the UK growing season is predicted to
decline and may make some non-irrigated crop production economically unsustainable. In addition the excessive
use of soil water by non-irrigated crops reduces the amount of rain water returned to surface or ground water
reserves. Irrigated crops will also require a greater input of supplementary irrigation to maintain production
because of both reductions in rain-fall and increases in evapotranspiration as average temperatures increase.
Consequently, there is a need to minimise the amount of water required to grow crops, and to this end it will be
important to have crop varieties that can use the available water more efficiently.
In addition to water, most crops require high inputs of inorganic fertilisers to maintain yield and quality. These
fertilisers are energy intensive to produce and can (if not correctly managed) pollute adjacent environments
leading to a loss of biodiversity. Capture of nutrients from the soil is a key crop trait that impacts on the quantity of
fertilizer that needs to be applied to soil and the entry of nutrients into water courses. Development of crop
varieties that can maintain yields with lower fertiliser inputs and that recover more applied nutrients are key to the
development of low input agricultural systems.
Previous work (HH3501, WQ0119, HH3608TX, LK0979, IF0125, HH3608TX, WU0116), has identified QTL
associated with water-use efficiency in the Brassica oleracea genome and validated this in the substitution line
AGSL118, which differs from the recurrent parental line (A12DHd), by substitutions of genomic DNA from the
other parent (GD33) in linkage group, C1, C6 and C7. Several QTL have also been identified for various
measures of phosphorus use efficiency (HH3501) on linkage groups C3 and C7 of B. oleracea in a number of
substitution lines.
SID 5 (Rev. 3/06)
Page 3 of 6
In order to define the position of these QTL more precisely, back-crosses to the A12 parent have been performed
and stocks of F1 or F2 seed produced, depending on the lines. For example, previously AGSL118 was crossed to
A12, and the resulting line (BC1F1) was grown and allowed to self-pollinate to give the BC1F2 seed stock. The aim
of this project was to identify recombinants in the BC1F2 population of the cross A12 x AGSL118 using molecular
markers. A panel of homozygous recombinant lines would then be assessed for phenotype to establish a single
small mapping interval for each QTL.
Results and Discussion
Objective 1. Continue back-crossing Brassica oleracea lines to further reduce the size of substitutions
and improve the resolution of recently discovered QTL for water and phosphorus-use efficiency.
The line AGSL118 has lower WUE and PUE than A12, and thus the three introgressions (on C1, C6 and C7) in
this line must contain a locus or loci that control these traits. We aimed to separate the three introgressions and
screen for recombination within the introgression in linkage group 7. A total of 421 BC 1F2 plants from the
introgression line AGSL118 were sown in modules in the glasshouse. Ten plants of the parental line A12DHd
were also sown with each batch. Plants were sampled in batches of 96 for DNA extraction from the 1 st true leaf at
the 2nd true leaf stage (approx 4 weeks after sowing).
DNA was extracted in 96 well format by homogenization of leaf material in a tissue mill, followed by extraction
using a commercially available kit (Qiagen DNeasy 96). Extracted DNA was stored at -20oC. PCR amplifications
were made in 96 well format using the primer pairs for markers identified as close as possible to the proximal and
distal ends of the introgressed regions listed under Objective 2. Fluorescently labelled reverse orientation primers
were used to facilitate capillary electrophoresis genotyping (Fig 1). PCR amplifications for each primer pair were
performed separately and aliquots of the amplifications then pooled for each sample to allow for screening of
multiple markers from a single plate.
In initial screens, two classes of plant genotype were selected and retained for self pollination and seed
production (Table 1).
1) Single introgressions, where the 3 introgressions present in AGSL118 had been reduced down to just one,
either in the homozygous or heterozygous form. Four recombinants in the linkage group 7 introgression have also
been detected, in a background lacking the linkage group 1 and 6 introgressions, offering an opportunity to begin
resolving the region of interest.
2) Double introgressions, where combinations of 2 of the 3 AGSL118 introgressions have been inherited. These
lines will enable us to test for allele combination effects should the phenotype not be retained by any of the three
single introgression lineages.
Figure 1. Automated genotyping traces corresponding to heterozygote signals for two of the markers screened;
Ni4B10 (175bp and 192bp) and sora93 (445bp and 479bp). The A or G suffix indicates the trace associated with
either the A12DHd allele (A) or the GD33 allele (G).
SID 5 (Rev. 3/06)
Page 4 of 6
Table 1. Numbers of AGSL118 plants detected (from 421 individuals) in each of
the genotypic categories identified. G and A indicate homozygous GD33 and
A12DHd genotypes respectively. H indicates a heterozygous genotype.
Genotypes
Linkage
Linkage
Linkage
Number of Plants
Group C1
Group C6
Group C7
Single
G
A
A
3
Introgression
H
A
A
3
G
A
A
1
H
A
A
11
G
A
A
10
H
A
A
9
Double
G
G
A
1
Introgression
H
G
A
7
G
H
A
8
G
G
A
4
H
G
A
8
G
H
A
6
G
G
A
3
G
H
A
17
H
G
A
8
Total number of plants
99
Approximately 70 of the above plants, representing all genotypes, have been grown to flowering and BC 1F3 seed
will be collected within two months.
Objective 2. Develop new markers for improving the resolution of QTL associated with water and
phosphorus use efficiency
Based on the existing B. oleracea genetic map, a total of 17 potential markers spanning the map interval of the
introgressed regions for AGSL118 were screened on GD33 and A12DHd parental and BC 1F1 (heterozygous
AGSL118) DNA. Three of these markers were associated with linkage group 1, five with linkage group 6 and nine
with linkage group 7. Four markers shown to be polymorphic between the two parents and within the introgressed
GD33 regions of AGSL118 were selected for screening our BC 1F2 plants. (Table 2).
Table 2. Results of marker screening on GD33DHd, A12DHd and AGSL118 DNA. ? indicates that
further optimisation of the PCR assay is required in order to obtain conclusive results. Markers selected
for screening are highlighted in italics.
Size (bp)
Linkage
Map position
Within introgressed
Group
Marker
(cM)
region?
GD33
A12
1
mSN3734a
65
No
288
1
Ol10F11
66
No
148
1
Ni4B10
101
Yes
192
175
6
6
6
6
6
mCRABSCLAWa
028L01AT3a
BoAP1-c
mBRMS298a
Ca72
38
39
40
41
82
No
?
?
No
Yes
?
?
232
234
273
?
?
219
253
7
7
7
7
7
7
7
7
7
sore90a_125
mBRAS023a_11
mBRAS019a_11
mNA12F03a
mCB10299a
mBRMS296a
mFITO222c
mA48350b
sora93
?
3
4
8
12
12
14
14
15
No
Yes
Yes
Yes
?
?
Yes
Yes
Yes
206
151
312
139
?
409
263
479
128
197
114
297
139
?
385
259
445
SID 5 (Rev. 3/06)
Page 5 of 6
For linkage group C7 we were able to select two informative markers, one at either end of the introgressed
region, allowing us to detect recombinants within the interval. There are four more markers immediately available
to further characterise the selected plants for this C7 region.
Only one of the 5 selected C6 markers was found to lie within the introgressed GD33 region of AGSL118 when
genetically tested. This one marker, Ca72, has allowed us to make informed selections at his stage; however, to
allow further characterisation of this region the development of more suitable markers is required.
Only one of the 3 selected C1 markers was found to lie within the introgressed GD33 region of AGSL118 when
genetically tested. This one marker, Ni4B10, has allowed us to make informed selections at his stage. However, it
is noteworthy that the C1 introgressed region of AGSL118 is largely in common with the introgressed region of
AGSL101, currently being characterised by Prof. Bill Finch-Savage as part of a BBSRC funded project
(BB/E006418/1). The polymorphic markers and BAC sequence data developed in this BBSRC project could be
directly applicable, should phenotypic characterisation of lines with single introgressions point towards C1 as
containing our gene(s) of interest.
Future work
For AGSL118, lines containing single and double introgressions for the linkage groups 1, 6 and 7 now need to be
assessed for WUE and PUE. This will determine the introgression, or combination or introgressions, responsible
for the WUE and PUE effects. In order to fine-map the QTL, recombinations within that region need to be
selected, and phenotyped. Very closely linked markers would become tools suitable for brassica breeding
programs. Such markers could also then be placed on the physical map, and finally the causative gene(s)
identified by further marker discovery and fine mapping. Once a causative gene is known, it would become a
breeding target in non-brassica crops.
Similar work is required for other substitution lines, where the work is less advanced, e.g. where we now have
generated BC1F2 seed suitable for mapping of PUE effects within specific introgressions. Again, this will lead to
genetic markers that could be used to select for increased PUE in brassica species.
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.
N/A
SID 5 (Rev. 3/06)
Page 6 of 6