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SID 4


Annual/Interim Project
Report for Period 2nd year
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Project details
2. Project title
Crop improvement for resource-use efficiency
Dan McGonigle
3. Defra Project Manager
4. Name and
address of
contractor
School of Life Sciences (formerly Warwick HRI)
University of Warwick
Gibbet Hill Road
Coventry
Warwickshire
Postcode
CV4 8UW
5. Contractor’s Project Manager
6. Project:
Miriam Gifford
start date .................
01/10/2010
end date ..................
30/09/2012
This form is in Word format
and boxes may be expanded
or reduced, as appropriate.
SID 4 (Rev. 3/06)
WU0128
1. Defra Project code
Page 1 of 12
Scientific objectives
7.
Please list the scientific objectives as set out in the contract. If necessary these can be expressed in an
abbreviated form. Indicate where amendments have been agreed with the Defra Project Manager, giving the
date of amendment.
1. To improve the resolution of recently discovered QTL for water and phosphorus-use efficiency
in Brassica oleracea by the fine mapping of introgressions in substitution lines.
2. To identify new loci and alleles associated with water and phosphorus-use efficiency and root
architecture in a model plant genome by using the most recent advances in allelic association
mapping in Arabidopsis thaliana.
3. To validate QTL for PUE, WUE and root architecture by testing candidate gene alleles for
association with traits in Brassica crop species.
Summary of Progress
8.
Please summarise, in layperson’s terms, scientific progress since the last report/start of the project and
how this relates to the objectives. Please provide information on actual results where possible rather
than merely a description of activities.
Our society faces a major challenge in meeting the demands of an expanding and developing global
population. Global demand for natural resources is driving up the costs of energy and food. We have
recently experienced the ramifications of this for food costs in the UK and will see its impact with further
starvation in developing nations. A global population expansion from its current level of 7 billion to 9.5
billion in 2050 will further aggravate food shortages. It is challenging to meet this demand for food
production and it is particularly challenging to achieve this in a sustainable fashion as climatic conditions
change. Two key efficiencies that we need to target for crop improvement are water and nutrient use
because of the declining availability of key nutrients such as phosphorus, the energy required for
production of nitrogen fertilisers and the need to reduce nutrient leaching to water courses and alleviate
increasing pressures on fresh water resources.
A major part of our work has been to identify parts of the plant genome (genes) that improve the ability of
Brassica plants to use resources. For this we have been following two lines of work:
In the first strategy we have been investigating one Brassica plant line that was previously been found to
have improved water use efficiency and one Brassica plant line that was found previously to more
efficiently use phosphorus. We have been using ‘fine mapping’ to identify which specific Brassica genes
are responsible for controlling these improvements. Results so far suggest that two regions of the genome
interact in a novel way to affect water use; we are narrowing down on a different region of the genome that
is associated with improved phosphorus use.
In a complementary strategy we have been testing collections of Brassica plant relatives in a large field
trial to discover which Brassica varieties are most resource efficient (maintain high growth rates, high
water use efficiency and accumulate optimal levels of nutrients) and discover the genes that control this.
Results so far have shown a strong interaction between the level of phosphorus in the soil and leaf dry
weight, giving us insights into how biomass accumulation is controlled by the environment. We are now
testing which parts of the genome are associated with these measured traits.
In parallel to our work in Brassica we are carrying out similar work on the model plant weed Arabidopsis
thaliana. As well as assessing phosphorus, water and nitrogen use efficiency in a collection of Arabidopsis
plant relatives we have been analysing root size since this controls how much of the soil a plant can
explore to take up water and nutrients. We are comparing the Brassica and Arabidopsis findings since if
we can identify that the same genes work in both species we will have good evidence that the genes play
these roles in other crops such as wheat and rice; these will be good targets for breeding programs.
** See scientific summary of progress below.
SID 4 (Rev. 3/06)
Page 2 of 12
Progress report
Reported below is the progress in the second year of this project. The progress updates are
organised according to the three objectives; associated milestones in this period are noted.
Objective 1: To improve the resolution of recently discovered QTL for water and
phosphorus-use efficiency in Brassica oleracea by the fine mapping of introgressions in
substitution lines
QTL for WUE: SL118
Previously, phenotypic analysis of Brassica oleracea BC1F3 lines revealed a significant reduction
in the plant WUE due to two introgression regions of parental line GD33 on chromosome 1 and
7. The WUE of some of these lines was lower than the recurrent parent, A12, indicating that the
A12 alleles at these loci were increasing WUE. In order to further fine map the uncharacterised
regions on chromosome-7, new KASPar (Competitive Allele Specific PCR based on SNP
detection system) markers are being designed. In addition some more SNP based markers were
designed through sequencing of the transcriptome of AGSL118; this will enable characterisation
through ‘high resolution melt analysis’ or sequencing.
To further test the results obtained from the above-mentioned study, another experiment
was conducted to evaluate the WUE of BC1F3 lines having a GD33 introgression on either
chromosome 1 only, chromosome 7 only, chromosome 1&7 only or on chromosomes 1, 6 & 7.
Data was collected for 10 replicates of each line, and its analysis has found that a GD-33
introgression on chromosome 1 only (BAT011) or chromosome 7 only (BAT052) does not affect
the plant WUE. However, in the genotype BAT070 (GD-33 introgression on chromosome 1 & 7
only) there is a significantly reduced plant WUE (Fig. 1). This suggests that introgression of GD33 on both chromosomes i.e. 1 & 7 is necessary to lower the WUE, hence there is a close
interaction between the two introgressed regions present on different chromosomes; the
potential of an epistatic interaction is in the process of being assessed using statistical methods.
On the basis of these results another experiment has been planned which will involve
phenotyping various recombinant lines that have already been generated so that the causative
introgressions can be defined with greater resolution on chromosome 7.
QTL for PUE: SL134
We are continuing progress with fine mapping the introgression on AGSL134. Seeds from the
first backcross were sown under glasshouse conditions, plants selfed, and the F2 generation
harvested for genotyping to identify individuals with regions of interest. These individuals will
then be used to assess their phosphorus use efficiency under optimal and low P availabilities.
SID 4 (Rev. 3/06)
Page 3 of 12
Fig. 1. Plant water use efficiency of BC1F3 Brassica lines grown under glasshouse conditions. Note
a significant reduction (P<0.05) in WUE of BAT070 which contains introgressions of GD33 only on
chromosome 1 and 7.
Objective 2: To identify new loci and alleles associated with water and phosphorus-use
efficiency in a model plant genome by using most recent advances in allelic association
mapping in Arabidopsis thaliana
Collection of WUE and PUE trait data from more balanced diversity sets
In the last year to make better use of the now 1,300 241k SNP-genotyped accessions and to
reduce the effects of linkage disequilibrium we determined a more balanced population of 96
ecotypes. We collected data from a controlled environment cabinet data designed to yield new
phosphorus and water use efficiency data from these 96 ecotypes. The experiment was carried
out in a Saxcil cabinet in short day (10hr light, 250 μmol PAR m-2 s-1) conditions with 18C day
temperature, 20C night temperature, and a vapour pressure deficit of 0.7 kPa. Two levels of P
were used (0g/l and 1.35g/l). The individual replicates were placed in a randomised design in the
Saxcil cabinet. The following parameters were measured and calculated: above ground fresh
weight, above ground dry weight, projected leaf area, chlorophyll content, gravimetric water use
efficiency (WUEp), gravimetric transpiration (Ep,), specific leaf weight (SLW), and mineral
analysis of δ13C and δ18O, P, Ca, Mg, K.
Data from this experiment were analysed using REML (a generalisation of ANOVA
suitable for unbalanced data). The differences between cabinets, between replicates within a
cabinet, between boxes within a replicate and between plants within a box were taken as
random factors, together with an interaction term between genotype and cabinet. For the
mineral data and chlorophyll content, only the high phosphate treatment could be analysed, and
genotype was taken as the only fixed factor. For the other traits, fertiliser treatment, genotype
and their interaction were taken as fixed factors. In addition, for dry weight, gravimetric water
SID 4 (Rev. 3/06)
Page 4 of 12
use efficiency, water content, chlorophyll content and specific leaf weight a joint REML analysis
was carried out with the data on the Nordborg collection obtained in DEFRA project HH3608TX.
This joint analysis did not include the data from the low phosphate treatment from the current
experiment which was not felt to be sufficiently similar to be compatible. For these analyses,
experiment was also taken to be a random factor. For all the traits analysed, the effects of
genotype were highly significant (pvalue <0.001).
The genotype means from these analyses were subjected to genome wide association
mapping (GWAS) using the methods developed in DEFRA project AT0438. Briefly, each SNP
was tested for significance by performing a Kruskal-Wallis one-way ANOVA on the trait data,
using the SNP as the factor. No direct adjustment is made for population structure, instead the
patterns that the SNPs induce on the Arabidopsis lines is studied, following the approach of
Aranzana et al (2005). Approximate significance thresholds were obtained by analysing 100
sets of random data and observing the lowest nominal p value across the genome. Since we
use a non-parametric test, the exact distribution of the random data used has no effect on the
nominal p values generated. The 5% global significance threshold for the joint analyses was
found to be approximately a nominal p value of 7e-7, and that for the current experiment alone
approximately 1.3e-6. In parallel to this analysis we are analysing our data with a mixed model
approach implemented with the program EMMA (as used for GWAS on Arabidopsis data in
Atwell et al 2010).
Association mapping analysis has been performed on data from the cabinet experiment
described above (for WUE and PUE) and for root trait data from Kas2 and Col0 (for investigating
how roots respond to combinations of varying P and N (RUE)). To identify genes associated with
the SNPs we are using 20kb windows around each SNP hit (following Atwell et al 2010); we now
have lists of candidate genes for each trait. As part of selecting the best two alleles for each of
WUE, PUE, and RUE for association mapping in Brassica (using Brassica oleracea orthologs of
these genes) we are carrying out further analysis using the multiple methods described above to
increase the confidence of selecting SNPs as good candidates for experimental validation.
Selection of loci in Arabidopsis and Brassica will be completed in time for development and use
of markers in Brassica oleracea in the final year of the project.
Objective 3: To further validate and discover new QTL for PUE and WUE by association
mapping in Brassica oleracea
Seed for 98 available BDFFS lines were obtained from Graham Teakle at the Warwick Crop
Centre. A full scale field experiment was conducted from May-July 2011, to evaluate the WUE
and PUE in the 98 BDFFS lines with 4 replications, irrigation, and two phosphorus (P) levels
applied to soil plots (Fig. 2A&B). A second field trial with the same 98 lines was carried out on
the same plot between July-August 2011. This experiment consisted of the same 98 lines with
three replications; no further P-fertiliser was added in these plots. Subsequent testing showed
that the P levels between the two P sites were heightened at the time of the second trial due to a
combination of release of the P in the high N plot and P use in the low P plot. Thus we expect to
make clearer observations on the effect of P in the second field trial.
SID 4 (Rev. 3/06)
Page 5 of 12
Fig. 2A. Brassica diversity fixed foundation set (BDFFS) lines growing
under field conditions in the first field trial (University of Warwick,
Wellesbourne campus) during May 2011.
Fig. 2B. Brassica diversity fixed foundation set (BDFFS) lines grown in the
first field trial at the time of harvest during July, 2011.
On each of the two occasions, the trial was laid out as a split plot experiment. Each main
plot consisted of an adjacent pair of beds, each 1.8m wide and 45.9m long. The same land was
used on each of the two occasions, with the same randomisation of the fertiliser treatments at
the main plot level. Each main plot was split into 98 subplots, for the different brassica lines, and
four guard plots, one on each end of each bed. Each subplot comprised 0.9m of bed, planted
with 5 rows per bed at 0.3m spacing between plants both along and across the bed. The outer
two rows of plants on each bed were guards, leaving nine experimental plants per subplot. The
subplots were laid out as a directly-randomised row-column resolvable incomplete block design.
At the time of harvest, specific leaf area, dry leaf weight, fresh leaf weight, whole plant
fresh weight, whole plant dry weight, and chlorophyll content parameters were measured. Single
leaf samples were dried and ground in preparation for mineral analysis (to be carried out by the
service in the Warwick Crop Centre) and stable isotope analysis (to be carried out by the service
in the Australian National University). This key mineral and isotope data is expected to be
available in the next few months.
All 98 BDFSS lines will be genotyped using the Brassica molecular markers designed
from the Arabidopsis GWAS analysis described in Objective 2 and a large number of random
SNP markers obtained from the VeGIN project. Associations will then be tested for with the WUE
and PUE phenotype data collected in the two BDFFS field trials. Associated genes will become
candidates for marker-assisted selection in Brassica breeding programs.
SID 4 (Rev. 3/06)
Page 6 of 12
Preliminary analysis of trait data: We are in the progress of analysing the data on chlorophyll
content (Fig.3. A & B), specific leaf area (SLA) (Fig. 4. A & B), dry leaf weight, fresh leaf weight,
whole plant fresh weight and whole plant dry weight for both individual and combined field trial
data. Preliminary analysis of the first experiment found that leaf chlorophyll content was found to
be statistically significantly different (pvalue<0.013) according to the P level in the soil
(interaction between genotype and P level). The highest leaf chlorophyll content (9.08 ccm units
(on sqrt scale) was found for Cabbage Plants under high P conditions, and the lowest (1.75 ccm
units (on sqrt scale) for Ornamental Kale under low P (Fig.3. A & B). An interaction between
traits and P level was also found for SLA, above ground plant dry weight, leaf dry weight, and dry
weight per plant in this experiment at pvalue <0.05.
Data analysis from the second experiment so far has revealed significant interaction
differences between genotypes and different P levels for leaf area. The lowest leaf area from a
standardised leaf (1.34 cm2 (on a sqrt scale)) under low P level was recorded in Kale, and the
highest leaf area (19.16 cm2 (on a sqrt scale)) was recorded in Tronchuda Cabbage (pvalue
<0.04) (Fig.4. A&B). A significant interaction between the genotypes and P levels in the soil was
also found for leaf dry weight, although no interaction was found for dry weight per plant, leaf
chlorophyll contents or SLA.
Associated Milestones:
- Milestone 3ii.b. Seed from core Brassica oleracea collection and sown for field trial
obtained
- Milestone 3ii.c. Data from Brassica field trial (WUE & PUE) collected
SID 4 (Rev. 3/06)
Page 7 of 12
A
B
Fig. 4. Square-root transformed leaf chlorophyll contents (ccm units) of different
genotypes grown under two phosphorus fertiliser levels in the first field trial during MayJune, 2011. A: genotypes from 1-49; B: genotypes from 50-98. Results were analysed
using ANOVA and means were compared at pvalue<0.05. Floating bars show the LSD
value. * = lowest chlorophyll contents, Ornamental Kale on low P; ** = highest
chlorophyll contents, Cabbage Plants on high P.
SID 4 (Rev. 3/06)
Page 8 of 12
A
B
Fig. 4. Square-root transformed leaf area (cm 2) of a standardised leaf on each plant from
different genotypes grown under two phosphorus fertiliser levels in the first field trial during
May-June, 2011. A: genotypes from 1-49; B: genotypes from 50-98. Results were
analysed using ANOVA and means were compared at pvalue<0.05. Floating bars show the
LSD value. * = smallest leaf area, Kale on low P; ** = largest leaf area, Tronchuda
Cabbage on high P.
References
- Aranzana MJ, Kim S, Zhao K, Bakker E, Horton M, Jakob K, Lister C, Molitor J, Shindo C,
Tang C, Toomajian C, Traw B, Zheng H, Bergelson J, Dean C, Marjoram P, Nordborg M. (2005)
Genome-wide association mapping in Arabidopsis identifies previously known flowering time and
pathogen resistance genes. PLOS Genetics. 1: e60.
- Atwell S, Huang YS, Vilhjálmsson BJ, Willems G, Horton M, Li Y, Meng D, Platt A, Tarone AM,
Hu TT, Jiang R, Muliyati NW, Zhang X, Amer MA, Baxter I, Brachi B, Chory J, Dean C, Debieu
M, de Meaux J, Ecker JR, Faure N, Kniskern JM, Jones JD, Michael T, Nemri A, Roux F, Salt
DE, Tang C, Todesco M, Traw MB, Weigel D, Marjoram P, Borevitz JO, Bergelson J, Nordborg
M. (2010) Genome-wide association study of 107 phenotypes in Arabidopsis thaliana inbred
lines. Nature. 465: 627-31.
SID 4 (Rev. 3/06)
Page 9 of 12
Amendments to project
9.
Are the current scientific objectives appropriate for the remainder of the project? ................. YES
NO
If NO, explain the reasons for any change giving the financial, staff and time implications.
Contractors cannot alter scientific objectives without the agreement of the Defra Project Manager.
Progress in relation to targets
10. (a) List the agreed milestones for the year/period under report as set out in the contract or any agreed
contract variation.
It is the responsibility of the contractor to check fully that all milestones have been met and to
provide a detailed explanation when they have not been achieved.
Milestone
Number
7
8
Milestones met
Target date
Title
M3ii.b Seed from core Brassica
oleracea collection and sown for field
trial obtained
M3ii.c Data from Brassica field trial
(WUE and PUE) collected
In full
On time
31/03/2011
yes
yes
31/08/2011
yes
yes
30/09/2011
yes
yes
Complete 2nd year annual report
14
(b) Do the remaining milestones look realistic? ..................................................................... YES
If you have answered NO, please provide an explanation.
Publications and other outputs
SID 4 (Rev. 3/06)
Page 10 of 12
NO
11. (a) Please give details of any outputs, e.g. published papers/presentations, meetings attended during this
reporting period.
Papers:



HARRISON, E., BURBIDGE, A, OKYERE, J.P., THOMPSON A.J. and TAYLOR I.B.
(2011) Identification of the tomato ABA-deficient mutant sitiens as a member of the
ABA-aldehyde oxidase gene family using genetic and genomic analysis. Plant
Growth Regulation 64:301-309
ZHANG, K, HILTON, H.W., GREENWOOD, D.J. THOMPSON, A.J. (2011) A
rigorous approach of determining FAO56 dual crop coefficient using soil sensor
measurements and inverse modeling techniques. Agricultural Water Management
98:1081-1090
CARTER, A., GIFFORD, M.L. (2010) Understanding how nitrogen regulates
developmental mechanisms using systems biology. Aspects of Applied Biology 105:
175-182
Presentations at tech transfer events:




Hammond JP (2011) Presentation to delegation from Bangabandhu Sheikh Mujibur
Rahman Agricultural University on crop nutrition research at University of
Nottingham. 4 August 2011.
Hammond JP, Broadley MR, Dupuy L, King GJ, Shi L, White PJ (2011) Brassica
root traits for resource use efficiency. UK-Brassica Research Community Annual
Meeting, University of Nottingham, UK, 17 May 2011.
Graham NS, Hammond JP, Broadley MR, King GJ, (2011) Update on Brassica
Biofortification. UK-Brassica Research Community Annual Meeting, University of
Nottingham, UK, 17 May 2011.
Hammond JP (2010) Exploring diversity associated with Phosphate uptake in
Brassica diversity sets. Oilseed Rape Genetic Improvement Network (OREGIN) 8th
Stakeholder Forum meeting, Rothamsted Research, 16 November 2010.
Presentations at scientific events:








Hammond JP, White PJ, Broadley (2011) Natural genetic variation in root
architectural traits within Brassica napus. 7th International Conference on Structure
and Function of Roots. Novy Smokovec, Slovakia; 5-9 September 2011.
Thompson AJ (2011) Invited keynote speaker: Rank Prize Meeting on “Roots for
improving resource acquisition in crops”. Lake District, UK; April 2011
Thompson AJ (2010) Association of Applied Biologists ‘Water and nitrogen use
efficiency in plants and crops’. Dec 2010
Gifford ML (2010) Invited keynote speaker. Association of Applied Biologists ‘Water
and nitrogen use efficiency in plants and crops’. Dec 2010
Thompson AJ (2010) Invited speaker: 7th Applying Genomics to Canola
Improvement Workshop, Saskatoon, Canada. Dec 2010
Hammond JP (2010) Predicting crop phosphorus requirements and developing new
diagnostic tools. Nutrient Use Efficiency Symposium, University of Copenhagen,
Denmark, 8 November 2010.
Thompson AJ (2010) Invited speaker BioMer Workshop ‘Root targeted
biotechnology for abiotic stress alleviation’ Nutrición Vegetal CEBAS-CSIC, Murcia,
Spain. Oct 2010.
Hammond JP Broadley MR, White PJ, King GJ, Welham S, Mayes S (2010)
Identifying genes in Brassica rapa associated with growth under low phosphorus
availability using a genetical genomics approach. 4th International Symposium on
Phosphorus Dynamics in the Soil-Plant Continuum, Beijing, China, 19-23 September
2010.
(b) Have opportunities for exploiting Intellectual
Property arising out of this work been identified? ............................................................ YES
If YES, please give details.
SID 4 (Rev. 3/06)
Page 11 of 12
NO
The aim of this project is to produce tools and materials suitable for breeding new crop varieties that
have improved water and nutrient use efficiency. Therefore this project could lead to IP on genetic
loci and molecular markers in Brassica for the selection and breeding of varieties with improved
nitrogen and phosphorus use efficiency, improved water use efficiency and increased root
size/growth. As the results develop we will identify specific opportunities and we are well placed to be
able to identify outlets for this through our participation in e.g. the VeGIN project meetings.
(c) Has any other action been taken to initiate Knowledge Transfer?................................... YES
If YES, please give details.
Future work
12. Please comment briefly on any new scientific opportunities which may arise from the project.
Declaration
13. I declare that the information I have given is correct to the best of my knowledge and belief.
Name
Position held
SID 4 (Rev. 3/06)
Prof. Brian Thomas
Deputy Head of Department
Page 12 of 12
Date
30/09/2011
NO