General enquiries on this form should be made to:Defra, Science Directorate, Management Support and Finance Team,Telephone No. 020 7238 1612E-mail: research.competitions@defra.gsi.gov.uk

SID 5 Research Project Final Report

SID 5 (Rev. 3/06) Page 1 of 34

NoteIn 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.

This form is in Word format and the boxes may be expanded or reduced, as appropriate.

ACCESS TO INFORMATIONThe information collected on this form will be stored electronically and may be sent to any part of Defra, or to individual researchers or organisations outside Defra for the purposes of reviewing the project. Defra may also disclose the information to any outside organisation acting as an agent authorised by Defra to process final research reports on its behalf. Defra intends to publish this form on its website, unless there are strong reasons not to, which fully comply with exemptions under the Environmental Information Regulations or the Freedom of Information Act 2000.Defra may be required to release information, including personal data and commercial information, on request under the Environmental Information Regulations or the Freedom of Information Act 2000. However, Defra will not permit any unwarranted breach of confidentiality or act in contravention of its obligations under the Data Protection Act 1998. Defra or its appointed agents may use the name, address or other details on your form to contact you in connection with occasional customer research aimed at improving the processes through which Defra works with its contractors.

Project identification

1. Defra Project code IFO120

2. Project title

Grass and clover genetic improvement to deliver environmental benefits

3. Contractororganisation(s)

IGERPlas GogerddanAberystwythSY23 3EBCeredigionUK                         

54. Total Defra project costs £ 448,648(agreed fixed price)

5. Project: start date................ 01 April 2007

end date................. 31 March 2008

SID 5 (Rev. 3/06) Page 2 of 34

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 Summary7. 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.

This work entails the use of state of the art (non GM) approaches to the genetic improvement of grasses and forage legumes to reduce the environmental impact of productive livestock agriculture in the UK.The work is divided into four parts.

1. Reducing diffuse pollution of water and air from livestock systems.

We have focused on two main aims. In the first we have developed new grasses and clovers which are likely to increase the efficiency of nitrogen (N) use in the rumen of sheep and cattle. Imperfect synchrony of protein and carbohydrate supply in the rumen contributes to inefficient N use and increased pollution of both nitrous oxide and leachable forms of N. To improve this we have developed lines of perennial ryegrass (Lolium perenne L.,) the most important grass of temperate pastures which have been crossed with plants from a related genera, Festuca (Fescues). Hybrids have been produced which show reduced rates of protein breakdown and therefore have considerable potential to allow improvements in synchrony and hence increased efficiency of N use. At the same time we have developed populations of white clover (Trifolium repens L.), the major forage legume of UK pastures, with reduced crude protein content. These plants are agronomically superior and represent a complementary approach to synchronising carbohydrate and protein supply to the rumen, particularly when combined with high sugar grasses. Further work in this area has focused on red clover (Trifolium pratense L.) and birdsfoot trefoil (Lotus corniculatus). Red clover shows high levels of activity for the enzyme polyphenol oxidase (PPO). This enzyme is associated with protection of protein and hence potentially reduced nitrogenous losses from the silo. We have shown variation between red clover varieties in the field with respect to PPO activity and also that a low PPO line shows reduced protein content during ensilement. Birdsfoot trefoil contains secondary compounds known as condensed tannins (CTs) which reduce the rate of protein breakdown, protect against bloat and may also reduce methane emissions. We have used in vitro approaches to show that there is a high CT line that had low ammonia emissions associated with reduced protein degradation.The second main aim in this part of the work is to reduce the need for fertilisers (N and phosphorus (P)) by increasing the efficiency with which these are taken up and used by the plant. Several approaches have been taken. We have developed more phosphorus use efficient populations of white clover and also investigated the importance of associations between this species and symbiotic mycorrihizal fungi in increasing phosphorus efficiency (PUE). Ryegrass and fescue-ryegrass hybrids have been assessed for their N and P uptake and also their ability to reduce overland flow of P- a major cause of pollution of waterways. In the field, differences have been observed indicating the potential of selecting for crop morphotypes able to reduce overland flow. The hybrids used allow us to associate these differences to a

SID 5 (Rev. 3/06) Page 3 of 34

species or species combination. Similarly we have laid the foundation for the identification of quantitative trait loci(QTL) underpinning PUE and NUE in both white clover and perennial ryegrass through the analysis of mapping families of both these species in horizontal sand bed lysimeters. Ryegrass-fescue hybrids have also been used to identify genes from fescues with the potential to enhance PUE and NUE when transferred into ryegrass.

2. Developing the potential of grass/clover pastures for carbon sequestration

Carbon sequestration in grasslands is an important, but not well understood, aspect of climate change mitigation. We have developed approaches to identifying QTL for key sequestration related traits in perennial ryegrass in particular shoot: root ratio, carbon content of litter and rate of litter return. A number of QTL for these traits were identified using horizontal sand bed lysimetry and experiments established in the field to verify these.

3. Improving the stability and resilience of grasslands through the use of multi-species mixtures

Increasing biodiversity in agro-ecosystems is an important policy objective and the relationships between plant diversity, resilience, productivity and delivery of ecosystem services are key questions of modern ecology. We have shown that multi-species mixtures composed of different functional groups (two grasses and two legumes) can be more productive than monocultures in many environments. Through this work-package these studies have been extended to investigate the effects of multi-species mixtures of different composition on N leaching and soil quality. The experimental design used focuses on the effects of species evenness (relative abundance) on these important functional outcomes.

4. Enhancing adaptation to climate change through improved water use efficiency and drought tolerance

Predictions for the UK suggest that the productivity and quality of UK pastures are likely to be adversely affected by climate change, particularly by reduced rainfall in the summer. This work takes a number of approaches to combating this. Introduction of genes from the more drought tolerant fescue species into perennial ryegrass is demonstrated to be a viable approach to increasing drought tolerance. Similarly backcross hybrids between white clover and the related species Trifolium ambiguum are more drought tolerant than white clover and show increased water use efficiency(WUE)Root morphology is a common factor with respect to both water use and impacts on soil quality (see above). Differences in root growth under drought stress and under irrigated conditions have been observed in certain ryegrass-fescue genome combinations and the regions of the genome responsible have been identified. In this work we are also building on studies showing that white clover can have a beneficial impact on soil quality through an effect on soil porosity.

The work described forms part of a ‘pipeline’ translating fundamental understanding and genomics (largely BBSRC funded) through to the dissection of complex traits, as described here, through to the use of this understanding in the development of new varieties of grasses and forage legumes. From April 1st 2008 work leading directly to new varieties will be funded through Sustainable Livestock Production LINK projects with substantial industrial support. New varieties of grasses and clovers are then commercialised by Germinal Holdings Ltd. A significant programme of technology transfer is undertaken which also includes the Grassland Development Centre at IGER.

Project Report to Defra8. 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

SID 5 (Rev. 3/06) Page 4 of 34

any action resulting from the research (e.g. IP, Knowledge Transfer).

SID 5 (Rev. 3/06) Page 5 of 34

1. Reducing diffuse pollution of water and air from livestock systems

1(a) Genetic improvement of grass and clover composition to optimise nutrient use in the rumen

Workpackage 1A

Objectives(i) Using markers transfer fescue genes for reduced proteolysis into elite ryegrass

germplasm to improved N-conversion in the rumen and reduced N loss into the environment.

(ii) Produce new ryegrass populations that combine fescue genes for improved protein stability in order to determine whether the same effect is reproduced when transferred to ryegrass. To achieve this we will produce new tetraploid L. perenne x F. glaucescens hybrids.

BackgroundIn pilot studies, Festuca glaucescens (4x) was hybridised with Lolium multiflorum (4x) and subsequently the F1 backcrossed onto diploid L. multiflorum to produce a series of backcross 1 (BC1) (3x) plants, four of which demonstrated improved protein stability in simulated rumen-like conditions due to the inclusion of relevant F. glaucescens genes. In the current project, we backcrossed selected backcross 1 (BC1) plants having fescue-derived improved reduced proteolysis onto Lolium (2x) in order to reconstitute the normal Lolium chromosome complement (together with relevant introgressed Festuca-derived genes) and to eliminate any supplementary Festuca chromosomes that reduce forage quality. We used fescue-specific AFLP markers identified in the Festuca parent and absent in the Lolium to tag Festuca-derived genes for reduced proteolysis in screens of the subsequent backcross 2 (BC2) population. Ultimately we will transfer the F. glaucescens genes for reduced proteolysis into L. perenne as this would be the ryegrass species of choice for UK conditions. L. perenne and L. multiflorum share the same protein stability under rumen conditions, and therefore information generated from transfer of F. glaucescens genes into L. multiflorum will be directly transferable for crop improvement programmes involving L. perenne. The creation of new tetraploid F1 hybrids between L. perenne and F. glaucescens (Objective (iii)) will be the starting point of a new introgression breeding programme that will require two further years of backcrossing onto L. perenne to complete.

Methods and ResultsObjective (i). The objective was achieved. Four BC1 plants BC1 P193/206 genotypes 4, 7, 10, and 19 had the longest protein half-life (circa 4hrs) amongst the BC1 generation. GISH analysis confirmed that the BC1 plants comprised 2 genomes of Lolium and 7 chromosomes of F. glaucescens. The 4 BC1

plants were backcrossed onto diploid L multiflorum cultivars, Atalja, Trajan, Meribel, AberMario and AberEpic to produce diploid BC2 populations. GISH analysis confirmed the presence of F. glaucescens introgressions onto Lolium chromosomes amongst the BC2 and that otherwise the diploid Lolium genome had been restored.

Amplified Fragment Length Polymorphism (AFLP) markers were designed that were present in the F. glaucescens, the F1, the BC1 and BC2 plants containing Festuca-derived chromosomes or chromosome segments, but were absent in all the Lolium parents. Festuca-specific AFLP markers present in BC2

plants having only single introgressed Festuca chromosome segments could only be present on those segments and should they also contain genes for reduced proteolysis will co-localise with these and serve as markers for their use and incorporation in introgression mapping programmes. Five Festuca-specific AFLP markers ACA-AAG(140), ACA-ACA(307), ACA-AGG(204), ACT-AGA(234) and ACT-AGC(100) were recovered in the BC2. Two BC2 genotypes one containing Festuca-derived AFLP marker ACT-AGA(234) and the other ACT-AGC(100) demonstrated reduced proteolysis compared with their Lolium parents (circa 2hrs) which was consistent during 2 independent tests. These AFLP markers will serve as early indicators of the location of Festuca-derived genes for reduced proteolysis. They will also serve to identify co-localising SSR markers which will be the marker system of choice in future crop improvement breeding programmes. (Milestone 1).

Objective (ii). The objective was achieved. L. perenne Ba14049 (4x) was crossed onto two F. glaucescens genotypes and 15 F1 hybrids produced. These were subsequently established in the glasshouse for future screens for evidence of reduced proteolysis compared with the Lolium parent. F1

SID 5 (Rev. 3/06) Page 6 of 34

hybrids having reduced proteolysis will be backcrossed onto diploid L. perenne cultivars showing high water soluble carbohydrate (WSC) expression to optimise rumen nutrient use. AFLP and subsequent SSR markers developed in the programme described in Objectives (i) and (ii) will assist in the targetting of genes for reduced proteolysis over generations in the backcross breeding programme to be undertaken here. (Milestone 2).

Objective (iii) Develop white clover lines with lower crude protein content BackgroundVariation within white clover and associated material for lower protein content and higher WSC has been identified. Interspecific hybrids between white clover and Trifolium ambiguum (Caucasian clover), developed at IGER, have a CP content 14.2g/kg DM lower than white clover indicating a potential 8% reduction in loss of dietary N. The WSC of these hybrids is also 6g/kg DM greater than white clover. Unique non fixing inbred genotypes of white clover have been used to demonstrate the principle that material of lower leaf protein content shows much slower protein degradation in the silo. Following this we established that genotypic variation within elite gene pools of white clover is much greater than was previously thought. Initial crosses have been made to produce lines for further testing using in vitro rumen simulation.

Methods and Results Two white clover lines with 5-10% reduction in crude protein content in the leaf have been developed from initial assessments of protein content using the Bradford assay. In this project we have polycrossed plants of these lines to produce sufficient seed for future studies. This material and white clover x Caucasian clover hybrids which show a similarly low protein content will be assessed in terms of their impact on rumen nitrogen use. In vitro rumen simulation (RUSITEC system) will be employed to this end focusing on ammonia production. This will provide information on the potential of low protein clover, when fed with a high WSC grass, to alter nitrogen partition, thereby reducing N-emissions, and provide targets for future selection. (Milestone 3).

Objective (iv) Quantify benefits of enhanced PPO activity in red clover

BackgroundA better understanding of the factors that influence the action of PPO is important in order to control and manipulate its potential benefit in conserved forage. Previous research within LS 3646 identified highly significant seasonal and varietal differences in levels of PPO activity with red cloverGrasslands Pawera was identified as having relatively low PPO activity not only for basal and optimal levels but also for % utilisation. This coupled with high protein levels suggests that this cultivar may be less effective at protecting its protein via PPO activity than the other cultivars under study.

Methods and ResultsA preliminary investigation on the effect of red clover varieties with varying levels of PPO activity on amino acid production in red clover silage was carried out in micro-silo conditions. Samples of Six varieties of red clover were taken from replicated field plots. The samples were wilted, macerated, inoculated and packed into tubes which were sealed with air-locks. Protein breakdown was monitored after 3, 5 and 90 day ensilage by measuring free amino acid content using a modified ninhydrin colorimetric assay (Fig 1). Data for each sampling date were analysed separately using analysis of variance (Genstat release 10).

SID 5 (Rev. 3/06) Page 7 of 34

0

0.1

0.2

0.3

0.4

0.5

0.6

G Pawera Milvus AberRuby Britta Sabtoron Deben

Free

amin

o ac

ids

(mM

ols

in 2

0g S

ilage

)3 Day

5 Day

90 Day

S.E. = ±0.0486, ±0.0472 and ±0.0449 for 3, 5 and 90 day ensilement (respectively), error D.F. = 10

Figure 1. Free amino acid content of 6 red clover silages at 3, 5 and 90 days

No significant varietal differences (P>0.05) were detected between the amino acid content of the red clover silages after 3, 5 or 90 days ensilement. However, there was a clear trend with free amino acids increasing with time and it was of interest that G. Pawera (low PPO activity) demonstrated relatively low amino acid content. The results show that there is no simple relationship between low PPO activity and increased proteolysis in red clover silage. (Milestone 4). Objective (v) Produce lines of Lotus corniculatus and quantify impact on methane production

BackgroundBirdsfoot trefoil (Lotus corniculatus) has great potential for methane reduction from ruminants as well as the ability to increase protein use efficiency, but its use is currently limited by poor agronomic performance in the UK. Methane production from housed sheep fed forage diets that contain L. corniculatus has been shown to be lower than those on ryegrass based pasture associated, in part, with the presence of condensed tannin (CT) in Lotus foliage. Similar responses have also been found in dairy cows grazing Lotus. The action by which CT reduces methane production is not known, nor is the effect of different levels of CT in Lotus on methane production. Recent work at IGER has shown differences between varieties of Lotus corniculatus in CT content and also the extent of variation within varieties. Diverse germplasm is now available with CT content ranging from 20mg/g DM to >100mg/g DM for experiments to quantify effect of CT content on methane in combination with other forage species. Rhizomatous lines of L. corniculatus with considerably improved persistence and contribution to mixed swards have been identified.

Methods and ResultsGas analysis techniques were used to quantify the efficiency of protein utilisation of a number of varieties/selection lines of Lotus corniculatus previously quantified for their CT content using a high throughput method of condensed tannin analysis developed in conjunction with previous Defra funded studies (LS3643). This has enabled the condensed tannin content of leaf and stem of a wide range of germplasm to be quantified, revealing significant variation in tannin content (20mg CT/g DM to 100mg CT/g DM). In a gas production experiment, the variety with the highest CT level (A1/0528) produced least ammonia indicating the lowest level of protein degradation amongst the 10 varieties/selection lines tested.

SID 5 (Rev. 3/06) Page 8 of 34

Figure 2. Ammonia production from gas analysis experiments comparing Lotus varieties with different levels of condensed tannins.

Crosses between agronomically superior germplasm (which are rhizomatous) which has good dry matter yield and persistence the germplasm that has different CT levels has been carried out leading to the development of 8 selection lines. Plants derived from these crosses will in future studies be polycrossed in the glasshouse enabling further analysis of the relationship between CT level, methane production and rate of protein breakdown to be quantified. Selection lines will also be grown in soils of different nutrient status to identify Lotus with CT levels that are reliable. This will provide information on the relationship between tannin content and N utilisation, identify targets for further selection and will ultimately lead to the development of Lotus germplasm that combines agronomic traits ensuring good persistence and high DM yield within the sward with the appropriate levels of CT that will optimise nutrient use within the rumen. (Milestone 5).

Further analysis of agronomically superior germplasm of Lotus corniculatus and L. uliginosus was carried out in a low fertility environment at Bronydd Mawr Upland Research centre (Latitude 51'57.69N and Longitude 03'37.53W). The land is classified as being within a severely disadvantage area and ranges between 240-390 m.a.s.l . It is dominated by an acid brown earth soil of the Milford series overlying Devonian Red Sandstone. Previous experiments at upland sites have demonstrated good establishment but difficulty in maintaining the legume within swards under a range of managements, particularly when grown in combination with white clover. This lack of persistency seriously restricts the wider uptake of this potentially useful species. Most of the plant material used in previous studies has been of European or North American origin (poorly adapted to UK conditions) and investigations have taken place in conditions of relatively high fertility. This research took place in a low fertility upland environment and investigates the persistence of germplasm previously identified by LS 3643.The original experiment was initiated in 2005 under the auspices of LS 3646. Nine selections of lotus were sown at Bronydd, in an area of low fertility, to compare their survival and performance with two extreme morphological forms of white clover and a ‘no clover’ control. Each treatment was replicated three times in a randomised split-plot design (plot size was 3 x 2m) with clover treatment as main plots and lotus as sub-plots. This design was chosen as white clover is a mobile invasive species and operationally easier to handle as main plots. Each lotus treatment was sown with Meadow Fescue (cultivar Rossa) as a mixture at a seed rate of 5kg/ha and 15 kg/ha respectively and the white clover component was sown at a seed rate of 1.5 kg/ha. The experiment was sown at the end of July 2005 and was well established by autumn of that year.

SID 5 (Rev. 3/06) Page 9 of 34

Herbage sampling took place on two occasions each year in 2006 and 2007. The first harvest took place each year when the Lotus showed 10-20% heading. All harvests were taken using the Shanks reciprocating blade mower with a cut width of 0.71m and cut to a height of 5cm. The cut length was measured on each plot at each sampling date. Fresh weight samples were taken and dried in an oven at 80ºC for 18 hours to estimate % dry weight. Further sub - samples for botanical analysis were also taken and frozen in a deep freeze at approximately -20 ºC to be processed over the winter months. All statistical analyses were carried out using analysis of variance (Genstat release 10.0) and data for 2006 and 2007 are presented in Fig 3. Highly significant differences (P<0.001) were detected between the lotus treatments for total herbage production in 2006 and 2007. All selections persisted well into the 2nd year. However, two selections Lotar and Ober Staedter, were outstanding and were significantly higher yielding than all other selections. Although the clover companion had no effect (P>0.05) on total herbage production in 2006 a highly significant effect (P<0.001) was detected in 2007 (Fig 4). The larger leaved white clover ‘Katrina’ produced a high yielding assemblage without compromising Lotus production. Leaf and stem samples were taken during the summer of 2007 and immediately frozen to enable condensed tannin content of this germplasm to be quantified.

012345678

2006 2007

S.E. = ±0.227 and ±0.365 for total DM yield for 2006 and 2007 (respectively), error D.F. = 52

Figure 3. Total Annual DM yield (t/ha) of nine lotus selections (mean of two clover companions and a no-clover control). Bronydd Mawr 2006-2007.

0

1

2

3

4

5

6

AberAce Katrina No clover

Tota

l Yie

ld t

DM/h

a

S.E. = ±021, error D.F. = 15

Figure4. Total Annual DM yield (t/ha) of lotus with two clover companions and a no-clover control (mean of nine lotus selections). Bronydd Mawr 2007.

SID 5 (Rev. 3/06) Page 10 of 34

The results indicate that lotus can be successfully established in low fertility environments. Clear cultivar differences were detected with Lotar and Oberhaus Staedter being particularly productive in this environment. Unlike previous experiments at this site all cultivars demonstrated good persistence into the second year and the presence of a white clover companion causes a significant improvement in the performance of the system. This information will be linked with the detailed morphological measurements to help identify important selection criteria and progress towards the development of Lotus germplasm that combines agronomic traits. Thus, ensuring good persistence and high DM yield within the sward with the appropriate levels of CT that will optimise nutrient use within the rumen. (Milestone 5).

1(b) Reducing the need for fertiliser inputs to UK livestock agriculture by increasing the nutrient use efficiency of grasses and clovers

Workpackage 1B

Objective (i) Development of white clover lines with reduced phosphorus requirement

BackgroundVariation in the requirement of P fertiliser without compromising agronomic performance has been demonstrated in a range of novel IGER white clover varieties indicating the potential to significantly reduce P use and overland flow of P from grassland.

Methods and ResultsWhite clover lines requiring no addition of P fertiliser under many soil conditions are being developed. An experiment initiated in a previous Defra funded study (LS3646) was used to test the hypothesis that populations of white clover selected for varying levels of yield and association with Vesicular Arbuscular Mycorrhizal (VAM) will affect the development and persistency of white clover in diverse swards in a low fertility environment. The experimental area utilised a site of extremely low fertility created at Bronydd Mawr Research Station through the cessation of fertiliser input onto upland grassland over a 12-year period. Soil analysis at the site in 2004 showed P, K, Na, Mg and Ca contents to be 3.6ppm, 0.31 meq%, 0.16 meq%, 0.77 meq%, and 4.65 meq% respectively, and the pH to be 4.97

Six treatments, including controls (Table 1) were replicated four times in a randomised complete block design (plot size was 3 x 2m) under a cutting regime.

Table 1. Six treatments including controls representing a range of white clover with varying yield / mycorrhizal characteristics

Treatment Line Description1 24 High yield + High mycorrhizal association – Ac 4246 2 25 High yield + low mycorrhizal association – Ac 42563 30 Low yield + High mycorrhizal association – Ac 42714 13 Low yield + low mycorrhizal association – Ac 41555 Trifolium repens standard cultivar cv. AberCrest6 No clover control

The background diversity was created by using a general purpose meadow mixture containing 13 Broad-leaved species together with a mixture of native grass species sown at a seed rate of 40kg/ha. The white clover populations were sown into seedling trays in the glasshouse and transferred into the plots once they had attained a reasonable size at a plant density of 16 plants/m2. Establishment was extremely successful and productivity, stability and the diversity of the treatments were monitored during 2007 which represented the second harvest year of the experiment (Figure 5). Herbage sampling took place on two occasions during 2007 using a Shanks reciprocating blade mower with a cut width of 0.71m. The cut length was measured on each plot at each sampling date. Fresh weight samples were taken and dried in an oven at 80ºC for 18 hours to estimate % dry weight and sub -

SID 5 (Rev. 3/06) Page 11 of 34

samples taken and frozen for later botanical analysis. The two harvests were combined to produce annual DM yields. All analyses were carried out using analysis of variance (Genstat release 9.2).

0

1

2

3

4

5

6

7

Family 13 Family 24 Family 25 Family 20 Abercrest No clover

Tota

l Yie

ld t

DM/h

a

Cut 1 Cut 2 Total

S.E. = ±0.369, ±0.150 and ±0.439 for total DM yield for cut1, cut2 and total annual yield respectively, error D.F. = 15

Figure 5. Components DM yield (kg/ha) of white clover selections of varying yield and mycorrhizal association 2007

Significant differences were detected between treatments for total herbage DM yield for Cut 2 (P< 0.01) and for total annual herbage production (P<0.05). Where significant differences existed, Family 24 (selected for high yield and high mycorrhizal association) significantly out performed all other treatments. AberCrest represented a typical white clover variety that was not selected for low fertility conditions and was consistently low in production. A more detailed botanical analysis of the species present within the broad leaved component is being carried out. The results emphasise the efficacy of selecting for performance in low fertility environments and suggest that further selection within this population will lead to the development of white clover cultivars that combine agronomic traits with enhanced performance in low fertility soils. The combination of selecting for high yield and high mycorrhizal association produced a relatively high yielding assemblage without any loss of diversity. This material is likely to be the basis of a new variety ensuring good persistence and high DM yield. (Milestone 6).

Objective (ii) Assess N and P utilisation in field plots of diverse ryegrass, fescue and Festulolium

cultivars.

Background. Four replicate 10 x 3m field plots with lysimeters were established at IGER North Wyke designed to monitor water and nutrient run off and comprised cultivars of diploid perennial ryegrass cultivar AberStar and Italian ryegrass cultivar AberEpic, diploid meadow fescue cultivar Bf993, hexaploid tall fescue cultivar Dovey, and tetraploid ryegrass x fescue hybrid cultivars Prior (meadow fescue x perennial ryegrass) and FL99-1(Festuca glaucescens x Italian ryegrass). These vary in both shoot and root morphology. As cultivars with very diverse growth and phenology they were assessed for differences in N and P utilisation and thereby their potential for reduced fertiliser application. N and P surface run-off and through-soil leachate was assessed separately. Due to limited resources only one sampling date was used but was chosen to follow heavy rainfall when run-off would be expected to be high.

Methods and ResultsOn the 6th December 2007, samples were taken from the drainage pathways from field plots of the 6 cultivars. The two pathways were: 1) Surface (surface and down to ~ 5 cm), 2) Through flow (~5-~30cm). The data presented was a single mean (n=3-4). Total phosphorus (TP) and total oxidised

SID 5 (Rev. 3/06) Page 12 of 34

nitrogen (TON) are presented. The major difference between the pathways was the higher levels of TP in the surface compared to through-flow pathway and this was likely to be due to surface soil having higher levels of P than soil lower in the profile. Although concentrations of P were low, the through flow TP was slightly lower in the fescue-based cultivars than the ryregass. Surface flow TP was lower for the perennial ryegrass, and tall fescue cultivars and there was a significant reduction between the Italian ryegrass x F. glaucescens hybrid cultivar 99-1 compared with the Italian ryegrass cultivar AberEpic. (Milestone 7).

Through flow_TP

0

50

100

150

200

250

FL99-1 Italianryegrass

Meadowfescue

Perennialryegrass

Prior Tall fescue

Con

cent

ratio

n (u

g/L)

Surface flow_TP

0.00

0.50

1.00

1.50

2.00

2.50

FL99-1 Italianryegrass

Meadowfescue

Perennialryegrass

Prior Tall fescue

Con

cent

ratio

n (m

g/L)

Figure 6. Through-flow total phosphorus (TP) and surface-flow TP in field plots of ryegrass, fescue, and ryegrass x fescue hybrid cultivars.

The two Italian ryegrass-based cultivars AberEpic and Festulolium cv 99-1 gave the highest through-flow of TON whilst TON was lower but similar for the other 4 cultivars. Surface-flow of TON was lowest in the perennial ryegrass-based cultivars AberStar and Festulolium cv Prior. Surface TON was significantly greater over the Italian ryegrass plots.

SID 5 (Rev. 3/06) Page 13 of 34

Through flow_TON

0.000.100.200.300.400.500.600.700.800.90

FL99-1 Italianryegrass

Meadowfescue

Perennialryegrass

Prior Tall fescue

Con

cent

ratio

n (m

g/L)

Surface flow_TON

0.00.51.01.52.02.53.03.54.04.5

FL99-1 Italianryegrass

Meadowfescue

Perennialryegrass

Prior Tall fescue

Con

cent

ratio

n (m

g/L)

Figure 7. Through-flow total nitrogen (TON) and surface-flow TON in field plots of ryegrass, fescue, and ryegrass x fescue hybrid cultivars.

Objective(iii) Phenotyping retention efficiency of N and P in ryegrass and white clover mapping

populations grown in horizontal sand-bed lysimeters.

BackgroundTwo studies were conducted in IGER’s system of shallow gradient (3%) sand-bed lysimeters (Fig.8), developed in Defra Commission LS3648, enabling simultaneous phenotyping of 80 mapping family genotypes for N and P recoveries in herbage. Briefly, mini-swards (0.115 m2) were established in 1m lengths of plastic guttering, arranged such that nutrient solution flowing through each gutter could be collected at the point of outflow. A key feature of this system was its linkage to IGER’s flowing solution

SID 5 (Rev. 3/06) Page 14 of 34

culture (FSC) system, providing precise computerised control of nutrient concentrations in the inflow solutions.

Methods and Results White clover:Mini-swards of seventy-eight progeny and the two parental genotypes of the stem nematode resistance mapping family were established in gutters using stolon cuttings and transferred into the lysimeter system once canopy closure had been attained. Plants were not nodulated at this stage and the experimental period, lasting 5.5 months, was split into three phases according to the source of plant N. During the first month (Phase 1), N was supplied automatically as 500 μM NO3

- in continuously recirculating nutrient solution, with all other nutrients maintained as described by Clement et al., (1978). The NO3

- supply was subsequently terminated and swards inoculated with a mixture of five strains of Rhizobia. Phase 2, covering the ensuing period of nodulation and establishment of N2 fixation, was arbitrarily defined as one month, during which the growth requirement for N was met primarily by remobilisation. Thereafter (Phase 3), plants were assumed to be entirely dependent upon N2 fixation. N and P recovery was estimated from herbage cuts (to a height of 1.5 cm above surface of the sand), consisting primarily of petiole and leaflets, on four occasions; at the end of Phase 1 (H1), at the end of Phase 2 (H2), one month into Phase 3 (H3) and at the end of Phase 3 (H4). Herbage was analysed for total N by continuous flow mass spectroscopy (Twenty-twenty, Europa Scientific Ltd, Crewe, UK) linked to a C/N analyser (Roboprep CN, Europa Scientific Ltd, Crewe, UK), and for total P (IGER Standard Method STM 035). In situ measurements of nitrate interception efficiency under the steady-state NO3

- influx (500 µM) conditions maintained during Phase 1 were performed on successive days (Runs 1-6). In each case, interception of nitrate by each sward was calculated from the difference between the nitrate flux entering and leaving the gutters. Samples of outflow solution were collected from each gutter and NO3

-

measured by automated colorimetry (Henriksen and Selmer-Olsen, 1970). Transpiration rates per gutter were measured by weight loss on two occasions, to provide correction factors for use in the flux calculations.

Figure 8. Shallow-gradient sand-bed lysimeters (LHS) and sandbox mini swards (RHS) employed, respectively, for phenotyping whiteclover and perennial ryegrass mapping families.N and P recoveries in herbage cuts expressed on a sward area basis showed a two to threefold range across the 80 mapping family genotypes (Fig 9). The degree of variation in N recoveries differed depending upon whether N was acquired as nitrate or by N2 fixation (data not shown). As expected, both N and P recovery were highly correlated with herbage yield in terms of dry matter (Fig. 10), although rather less so in the case of P, indicating the possibility of decoupling the two traits.

05

1015

202530

35

<12 12-16 16-20 20-24 24-28 28-32 >32

Nos

gen

otyp

es

gN/m2

A

0

5

10

15

20

25

30

1 1.2 1.4 1.6 1.8 2 More

Nos

gen

otyp

es

gP/m2

B

SID 5 (Rev. 3/06) Page 15 of 34

Figure 9. Frequency diagrams for cumulative recovery of (A) total N and (B) and total P in herbage cuts from 80 whiteclover mapping family genotypes grown as mini-swards in shallow gradient lysimeters over 5.5 months. Data are for H1-H4 in the case of N and for H1-H3 in the case of P.

R2 = 0.86

10

15

20

25

30

35

200 300 400 500 600 700 800 900

gN/m

2

Herbage dry weight (g/m2)

A

R² = 0.65

0.5

1.0

1.5

2.0

2.5

200 300 400 500 600 700

gP/m

2

Herbage dry weight (g/m2)

B

Figure 10. Relationships between cumulative (A) N and (B) P recoveries and herbage yields across 80 whiteclover mapping family genotypes grown as mini-swards in shallow gradient lysimeters. Data are for H1-H4 in the case of N and for H1-H3 in the case of P. R2 values refer to linear regressions of data.

Whilst there was some agreement (r2 = 0.59) between the ranking of genotypes for cumulative N and P recoveries (Fig. 11A), the occurrence of significant variation in P recovery for a given N recovery and vice versa suggests that co-selection for these traits may not be obligatory.

R² = 0.59

5

10

15

20

25

0.5 1.0 1.5 2.0 2.5

N re

cove

red

(gN

/m2)

P recovered (gP/m2)

A

01020304050607080

0.5

1.5

2.5

3.5

4.5

5.5

6.5

7.5

8.5

Mo

…Num

ber

ofob

serv

ation

s

Nitrate interception rate (kgN/ha/d)

B

Figure. 11. Relationships between (A) cumulative N and P recovered in herbage cuts H1-H3 by 80 whiteclover mapping family genotypes grown as mini-swards in shallow gradient lysimeters (R2 values refer to linear regression of data), and (B) the frequency distribution of nitrate interception rates by 80 whiteclover genotypes measured during Phase 1 (Totals for Runs 1-6).

The combined data from the in situ measurements of nitrate interception efficiency during phase 1 (Fig. 11B) indicated a five-fold variation in maximum rates of nitrate capture across the mapping family in the absence of N2 fixation. (Milestone 8).

Perennial ryegrass.N recoveries by mini swards of 94 progeny and the two parental genotypes of the amenity x forage Lolium perenne mapping family were measured in replicated sand-box mini-swards (surface area 900 cm2), established in 2004 (Fig. 8) during a previous Defra Commission (LS3648). Irrigation was applied during drought periods and nutrients applied as a combination of slow-release (Polyon 18-6-12+Tr) and soluble (Phostrogen 14-10-27 plus trace elements) fertiliser in five split dressing giving a total application of 600 kgN/ha for 2004, 336 kgN/ha in 2005, 184 kgN/ha in 2006 and 393 kgN/ha in 2007. A range of trait-assessments were performed between July 2004-July 2007 in accordance with Defra project LS3648. However, these did not extend to determining apparent N recoveries. Consequently herbage samples from harvest cuts (to a height of 3 cm above sand level) taken on seven occasions (13/07/04, 15/09/04, 5/05/05, 15/06/05, 20/07/05, 30/09/05, 06/07/06) were analysed for total N and P as described above for whiteclover. Apparent fertiliser N recoveries in herbage cuts 1 and 2 varied five fold across the mapping population grown as mini-swards in sand-boxes (Fig. 12A). The relatively low absolute recovery rates reflect the susceptibility of the sand medium to nitrate leaching losses. Absolute herbage N recoveries estimated

SID 5 (Rev. 3/06) Page 16 of 34

from the cumulative N content of harvests 1-6 were correlated reasonably well (r2 = 0.59) with the initial fertiliser N recoveries (Fig. 12B) suggesting in the first instance that phenotyping for this trait does not necessarily need to extend across the entire growing season.QTLs for apparent fertiliser recovery (H1 & H2) and cumulative N recovery (H1-H6) were identified (Table 2) on the two AFLP-based outline maps (amenity alleles and forage alleles), both with seven linkage groups, produced for Defra LS3648, by interval mapping and Kruskal-Wallis nonparametric single-locus analysis, applied to all mapped and unmapped markers using MapQTL Version 4.0.

0

4

8

12

16

20

5 10 15 20 25 30 35 40 45 50 55 60Fertilizer N recovery (%)

Num

ber o

f gen

otyp

es A

R2 = 0.59

0

50

100

150

200

250

300

0 10 20 30 40 50 60Fertilizer N recovery (%)

Tota

l N re

cove

red

(kgN

/ha)

B

Figure 12. (A) Frequency distribution for apparent fertiliser N recoveries in herbage cuts 1 and 2 by 94 progeny and two parental genotypes of the amenity x forage Lolium perenne mapping family grown as mini swards in sand boxes, and (B) the relationship between Total N recovered in herbage across cuts 1-6 and apparent fertiliser N recoveries based on cuts 1 and 2. Data are means of two replicate swards per genotype. The QTL at the top of (forage alleles) linkage group 1 (Locus: f_ACT-CTA.187) appears particularly promising and clearly merits further confirmation. (Milestone 8).

Parental allelic Trait Linkage Position LOD % variance Locus Kruskal-Wallisorigin group (cM) score explained Significanceforage fertiliser N recovery H1&H2 (%) 1 0 4.2 18.6 f_ACT-CTA.187 0.0001

fertiliser N recovery H1&H2 (%) 1 9.7 2.19 10.2 f_ACC-CAA.64 0.005fertiliser N recovery H1&H2 (%) 1 12 2.22 10.3 f_ACA-CAC.37 0.005fertiliser N recovery H1&H2 (%) 1 14.4 1.95 9.1 f_ACC-CCC.91 0.005

forage Cumulative N recovery H1-H6 (kgN/ha) 1 0 4.4 19.4 f_ACT-CTA.187 0.0001Cumulative N recovery H1-H6 (kgN/ha) 1 22.9 2.4 11.1 f_ACC-CAC.84 0.001

amenity Cumulative N recovery H1-H6 (kgN/ha) 1 42.8 2 9.3 a_ACT-CAA.161 0.005Cumulative N recovery H1-H6 (kgN/ha) 1 44.7 1.91 8.9 a_ACTCTC.207 0.005Cumulative N recovery H1-H6 (kgN/ha) 3 21.2 2.14 9.9 a_ACT-CTA.193 0.005

Table 2. QTLs for apparent fertiliser N recovery (H1 & H2) and cumulative N recovery (H1-H6) on the two AFLP-based outline maps for the amenity x forage Lolium perenne family. The recorded QTL position is that of the peak LOD score given by interval mapping. The significance level of the Kruskal-Wallis test statistic associated with each of the QTLs is also given. % variance explained = percentage phenotypic variation explained by the QTL.

Objective (iv) Locating Festuca genes for NUE and PUE in Lolium x Festuca introgression lines

BackgroundResults from a preliminary characterisation of growth, nitrate uptake and leaf N productivity by a range of Festuca-lolium chromosome substitution lines conducted in flowing solution culture suggested Festuca-derived Chromosome 3 was associated with high rates of nitrate uptake, whilst Chromosome 4 was associated with high N productivity. As a first step in identifying the location of the responsible Festuca genes, a range of substitution lines containing different fragments of chromosome 3 were phenotyped as mini-swards in the system of horizontal sand-bed lysimeters described under WP1B (iii) above.

SID 5 (Rev. 3/06) Page 17 of 34

Eighteen different Festuca x Lolium Chromosome 3 substitution lines plus 11 additional parental genotypes and assorted substitution lines were established as mini-swards (0.115 m2) in 1m lengths of plastic guttering. Two replicate gutters of each of the Chromosome 3 substitution lines, and four replicates of each of the 11 additional genotypes were prepared, giving a total of 80 gutters. Following transfer into the system of horizontal sand-bed lysimeters interfaced with IGER’s Computer-controlled flowing solution culture system, the gutters were subjected to regular ‘feeding’ by flushing through with recirculating nutrient solution, on a cycle of one day ‘on’ and three days ‘off’, over the next six weeks to promote the development of a dense sward. Herbage yields, N recovery and nitrate interception efficiencies were subsequently measured over one month, during which the pH and composition of the recirculating nutrient solution was automatically controlled as described for Phase 1 of the whiteclover study in WP1B (iii) (i.e. at 500 µM NO3

-). Swards were cut to a height of 6 cm above the sand surface at the start (H1) and end of this period (H2) to assess yield, total leaf area and N recovery (gN/m 2

ground area). Nitrate interception efficiencies (see WP1B(iii)) were measured under the steady-state NO3

- influx (500 µM) conditions on ten occasions (Runs 1-10) and transpiration rates on two occasions to enable nitrate influx to each gutter to be more accurately estimated from the measured solution outflow rates.

Methods and ResultsThere was substantial variation amongst the Chromosome 3 substitution lines in terms of N recovered in the herbage increment between cuts 1 and 2 (Fig. 13A), the range being 1.5-4.8 gN/m2. The majority of this variation was associated with yield differences (data not shown) rather than differences in %N in the herbage dry matter (Fig. 13B), which ranged between 3.1- 4.5%. Interestingly, the Chromosome 4 substitution reference genotype (Chr 4 Bx468.64) recovered 6.7 gN/m2, exceeding all Chr. 3 sub-lines.

Bx462.925

Bx462.926

Bx462.927

Bx462.822B

x462.83B

x462.929B

x462.930B

x462.931B

x462.933B

x462.806B

x462.820B

x462.843B

x462.848B

x462.920B

x462.921B

x462.922B

x462.923B

x462.924B

x469.57

0.0

1.0

2.0

3.0

4.0

5.0

6.0

Nitr

ogen

reco

very

(gN

/m2 )

Chromosome 3 substitution line

A

Bx462.925

Bx462.926

Bx462.927

Bx462.822B

x462.83B

x462.929B

x462.930B

x462.931B

x462.933B

x462.806B

x462.820B

x462.843B

x462.848B

x462.920B

x462.921B

x462.922B

x462.923B

x462.924B

x469.57

0.0

1.0

2.0

3.0

4.0

5.0

6.0

%N

in h

erba

ge

Chromosome 3 substitution line

B

Figure 13. (A) Variation in N recovery in herbage cut 2 by selected Chromosome 3 substitution lines and (B) %N in the herbage dry matter of cut 2. Values are means (n=2) ±SD.

Nitrate interception efficiencies measured on a mini-sward basis, and expressed for ease of comparison as maximum nitrate uptake rates per sward (Fig. 14A) showed a similar pattern of variation across the 18 Chr. 3 sub-lines assessed to that for N recovery, with mean values ranging from 0.8-3.1 µmoles NO3

-/minute. The inter-chromosomal comparisons (Fig. 14B) confirmed the superior performance of the Chr. 4 sub-line (Chr 4 Bx468.64) and indicated significant transgressive variation with respect to the performance of several sub-lines compared with the parental genotypes. In conclusion (1) reasonable agreement was observed between the herbage analysis and nitrate interception measures of N recovery in terms of ranking of genotypes and (2) potentially useful variation was revealed amongst the Chr. 3 sub-lines assessed. However, the superiority of the Chr.4 sub-line (Chr 4 Bx468.64) suggests that detailed assessment of the role of Ch.4 in conferring enhanced NUE is advisable. (Milestone 9).

SID 5 (Rev. 3/06) Page 18 of 34

Bx462.925

Bx462.926

Bx462.927

Bx462.822B

x462.83B

x462.929B

x462.930B

x462.931B

x462.933B

x462.806B

x462.820B

x462.843B

x462.848B

x462.920B

x462.921B

x462.922B

x462.923B

x462.924B

x469.57

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

Nitr

ate

upta

ke (µ

mol

es/m

in A

Chr 1 Bx468.83

Chr 2 Bx469.78

Chr 3 Bx469.57

Chr 4 Bx468.64

Chr 5 Bx470.102

Chr 6 Bx468.208

Chr 7 Bx471.5F1 Bx467.2

LipriorM

eltra

F. pratensis

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

Nitr

ate

upta

ke (µ

mol

es/m

in) B

Figure 14. Nitrate interception efficiencies for (A) selected Chromosome 3 substitution lines and (B) 11 additional parental genotypes and assorted substitution lines, expressed as maximum nitrate uptake rates per mini-sward (0.115 m2 )grown in 1m lengths of shallow guttering. Values are means of Runs 1-10. Error bars are ±SD

Objective (v) Modelling analysis of environmental benefits resulting from ryegrass and clover with

improved NUE and PUE

BackgroundConsiderable genetic improvement in nitrogen-use-efficiency (NUE) has been demonstrated in new lines of perennial ryegrass (>30%), as has the knowledge of the location of those genes responsible and of the gene markers suitable for their selection and use in cultivar development. Compared with low NUE-selections, high NUE-marker-selections produced only 1% less dry matter with a 50% reduction in fertiliser rate, and had 4% lower forage N content. Computer modelling has demonstrated that grassland economic sustainability is sustained given a 10% improved NUE and 45% reduction in fertiliser application. This combination achieves targets that meet the EUNO3 Directive for water quality and leads to a 36% reduction in P leaching. Pilot studies have indicated that fescue species are more nutrient-use-efficient than ryegrass and that the trait is readily transferable to ryegrass.

Methods and ResultsProgress in increased NUE, reduced fertiliser use, lower dietary intakes of N and P etc and improved nitrogen conversion in the rumen through use of high sugar grasses or those with reduced proteolysis, will result in reduced greenhouse warming potential (including methane) and acidification potential as well as reduced diffuse N and P pollution of waterways. This was clearly shown using the SIMSDAIRY modelling approach. This demonstrated significant benefits with respect to air quality where improved NUE was combined with reduced fertiliser applications. N2O was reduced by 57%, and ammonia by 20% when N plant absorption increased by 10% and was combined with a 45% reduction in fertiliser application, all with no loss of farm economic sustainability. (Milestone 10).

Implications and future work (Workpackage 1A)Implications of main findingsWe have developed white clover selection lines and interspecific hybrids between white clover and Caucasian clover that have lower crude protein content than white clover, providing material that will form the basis of future white clover varieties. This material will also enable us to test the hypothesis that white clover with a lower protein content in mixtures with a high WSC grass, will improve the efficiency of N utilisation in the rumen and reduce N pollution. We have also shown, using gas analysis techniques, that selection lines of Lotus corniculatus that differ in condensed tannin content also differ in ammonia production confirming a relationship between CT content and protein utilisation. Information has also been gained on the persistence and productivity of selection lines of Lotus corniculatus in plots and spaced plants providing genetic material for future selection and analysis. In red clover the investigations into field activity of PPO showed that there is no simple relationship between low PPO activity and increased proteolysis in red clover silage and clearly further study is required.Existing backcross 1 (BC1) plants including Festuca glaucescens genes for reduced proteolysis were backcrossed onto Lolium multiflorum and diploid backcross 2 (BC2) plants were identified containing single introgressed fescue-derived chromosome segments. The presence of the single introgressed

SID 5 (Rev. 3/06) Page 19 of 34

fescue chromosome segments was confirmed by GISH. Fescue-derived AFLP markers (the markers of choice in preliminary introgression studies as the provide for wide genome coverage) were identified amongst the BC2 and must co-localise with the identified GISH-labelled introgressed fescue chromosome segments. Significant improvements compared to the Lolium multiflorum parents in protein stability were found in some of the BC2 plants with fescue chromosome segments demonstrating the heritability of the trait. Despite an improvement in reduced plant-mediated proteolysis in the BC2, protein stability was reduced significantly from that recorded previously in the BC1 parents.New hybrids involving L. perenne (the ryegrass of choice for UK conditions) and F. glaucescens were produced, but the hybrid combination is more difficult to achieve than with L. multiflorum.Future workWe will use gas analysis techniques to quantify the impact of low protein clover on protein utilisation in comparison with existing varieties and in mixtures with perennial ryegrasses with high WSC content. We will use this information to provide targets for future selection of varieties. The stability of CT content of the best material of Lotus corniculatus identified in IFO120 will be quantified across different environments and soil types. This information combined with analysis on the survival and persistence of Lotus corniculatus will be used for further crosses to develop advanced selection of Lotus corniculatus that will be the basis of future varieties.More fundamental research is required to understand the genetic control of the reduced-proteolysis trait found in F. glaucescens. The reduction in expression of the trait amongst the BC2 compared with the BC1 generation would suggest an additive QTL gene effect with the genes responsible dispersed across the genome. Gene expression may be enhanced through identifying the locations of the primary genes for the trait and subsequently producing homozygous lines. An introgression-breeding approach is well suited to achieve this objective. However, it may well be found that an amphiploid approach will be necessary which will require producing stable Festulolium hybrids that comprise entire genomes of Lolium multiflorum or L. perenne together with F. glaucescens in order to achieve the necessary enhanced protein stability of ryegrass when ingested in the rumen.

Implications and future work (Workpackage 1B)Implications of main findingsWe have also gained information on persistence and productivity of white clover selections that perform well under low fertility conditions and identified selections that will provide material for future development.Total phosphorus and total nitrogen leaching was compared both through and over soil surfaces of established field-plots at Rowden North Wyke containing diverse grass cultivars with different plant morphology (both foliage and roots). Passage overall of Total P was greater across the plot surfaces than through the soils and was less generally with plots involving the tall fescue cultivar or the Festuca glaucescens hybrid. Through soil flow of N was greater than the surface flow for all species cultivars but tall fescue was again the most efficient species cultivar at capturing and restricting loss of N.We have identified a number of provisional QTLs for N fertiliser recovery in perennial ryegrass which, subject to confirmation in elite agronomic germplasm, may facilitate marker assisted selection for this trait. Likewise, the variation demonstrated amongst the Festuca x Lolium Chromosome 3 substitution lines for N recovery and nitrate interception efficiencies will enable precise targeting of the Festuca chromosomal regions for incorporation in hybrids designed for enhanced NUEFuture workBoth foliage morphology, growth habit, and persistency and root growth especially at times of heavy rainfall will affect the amounts of leachate of N and P. Detailed research is planned in Defra Commission work IFO145 to monitor both movement and use of water and P and to identify foliar and rooting traits best able to more efficiently achieve improved water-use-efficiency and to better ensure high water quality. We will characterise Festuca x Lolium Chromosome 4 substitution lines for N recovery and nitrate interception efficiencies and determine the extent to which QTLs for different components of NUE in perennial ryegrass, including fertiliser N recovery, are specific to level (high v. low) and form (i.e. NO3- or NH4+ ) of N supply. This information will be used to develop ‘proof of principle’ populations exhibiting high NUE specific to form and plane of N supply.

2. Developing the potential for grass/clover pastures for carbon sequestration

SID 5 (Rev. 3/06) Page 20 of 34

Workpackage 2

Objective(i) Identification of QTLs for traits associated with C sequestration in perennial ryegrass BackgroundEnhancing C sequestration through genetic improvement of forage species would provide a low-cost addition to the current options for increasing C storage in grassland systems, which focus on conversion from temporary to permanent grassland and alterations in N input (i.e. Soussana et al., 2004). Direct comparison of rates of carbon sequestration by different genotypes requires measurement of SOM levels under field conditions over many years. However, candidate traits likely to affect C sequestration are identifiable from experimental and modelling studies and are classifiable according to whether they affect (a) quantity and mass transfer (e.g. primary productivity, shoot;root ratio, litter production, root turnover, root exudation), (b) quality (e.g. litter C:N ratio, lignin content, N resorption efficiency), or (c) placement and risk of decomposition and loss (e.g. rooting depth). Work in the present project concentrated on identifying QTLs for shoot:root ratios and litter production using existing mapping populations and experimental systems developed in Defra LS3648, together with initiating a field study of intra-specific variation for a wider range of traits likely to affect C storage in the soil profile, the initial results from which are currently being analysed and are therefore not reported here.

Methods and ResultsQTLs for shoot:root dry weight ratioScreening of 94 progeny genotypes plus the two parental genotypes belonging to IGER’s amenity x forage type Lolium perenne mapping family for variation in shoot:root dry weight ratios under different nutritional conditions was undertaken in flowing solution culture during a previous Defra Commission (LS3648), and a number of QTLs identified on a pair of AFLP-based outline maps. In order to examine the robustness of these QTLs, two further screens were performed using identical experimental protocols to those employed in LS3648 and the entire data set remapped onto an alternative microsatellite based map, allowing direct cross-referencing with other Lolium maps.QTLs for litter return The occurrence of QTLs for C return in litter (primarily senescent leaves and stems) was investigated by two approaches. The first consisted of reworking data obtained for 94 progeny and the two parental genotypes of the amenity x forage Lolium perenne mapping family, grown in sand-box mini-swards, during a previous Defra Commission (LS3648), in the form of herbage yields and accumulated surface litter accumulated during the 2006 growing season. The data were re-mapped onto a microsatellite based map (79 genotypes) constructed for the amenity x forage Lolium perenne and the positions of QTLs for the gross C contents of the herbage and litter compared.In a second approach, net litter production was assessed in mini-swards of 78 progeny and the two parental genotypes of the amenity x forage Lolium perenne mapping family maintained in shallow gradient lysimeters (i.e. 1m sand-filled gutters) under ambient greenhouse conditons. These swards had previously been used for measurements of plant N relations during 2006 (Defra LS3648) in IGER’s system of shallow gradient sand-bed lysimetry (Fig.1), before transfer to an ambient greenhouse in December 2006. Throughout 2007 they were subject to regular cutting (to 6cm height), liquid fertiliser application and watering to maintain sward quality. Net litter production by each sward during 2007 was measured on 5/11/7, using a steel comb to remove all detached senescent leaf and stem material from the surface of the gutters. Samples were oven dried for 24 h at 80oC before weighing and grinding for analysis of Total C and N as described under WP1B (iii) above.Field evaluation of intra-specific variation in traits associated with carbon sequestration in perennial ryegrass. An existing series of field-plots was utilised in Cae Penrhyn, IGER Aberystwth, established in June 2006 and consisting of replicated 1 m2 plots, including eight varieties of perennial ryegrass (Aberimp, Ace, Ba13516, Bargold, Bizet, Brightstar, Cadix, Greenfair). Half the plots received zero N fertiliser and half received 360 kg N/ha/year, plus background nutrients in 6 applications. Prior to this study all plots were cut once a week throughout the growing season to a height of 30mm, with the clippings removed. Trait assessments were performed over a three month period (15/8/7 – 16/11/7) period, between 16 July 2007 and 24 September 2007. All plots were cut (to 2 cm height) immediately prior to the start of this period and again on 12/11/7 to measure above ground productivity. Herbage was weighed and dried, and subsamples ground for total N and C analysis as described under WP1B (iii) above.

SID 5 (Rev. 3/06) Page 21 of 34

Standing litter was removed from duplicate fixed location 15 cm x 15 cm quadrats within each plot with a steel comb at the start of the, and subsequently on a three-weekly basis throughout the experiment. All litter fractions were oven dried, weighed and ground for analysis of total C and N as above. Leaf turnover was measured using a standard leaf marking technique on ten tillers per plot, with leaf counts made on a fortnightly basis. Following the final herbage cut, duplicate soil cores (0-10 cm depth, 5.7 cm diam.) were removed, fresh weight bulk densities measured, a representative soil subsample (2g) taken for determination of soil moisture content and total C, and associated plant material washed out of the cores with tap water and separated into two fractions: (a) Live leaf/shoot, and (b) live /dead roots, litter and course organic matter and detritus, for total C and N analysis.The majority of the QTLs (LODs 2.04-4.53) for shoot:root dry weight ratios of perennial ryegrass were unique to a specific environmental (i.e. nutritional) condition, although three of the QTLs assigned to linkage group 7 (Loci: LpACA24B2(2), LpHCA18F11(7), LpHCA18F11(6)) were common to two different nutritional conditions. Whilst this suggests strong G x E effects, the occurrence of useful generic QTLs controlling shoot:root ratios cannot be ruled out at this stage. Several QTLs for both herbage yields and accumulated surface litter were identified from the sand-box experiment (Table 3). These were predominantly on linkage group 7 and covering similar regions of map. Whilst this was not unexpected given that yields of herbage and litter are inextricably linked, the results indicate a potentially useful degree of exclusivity in terms of markers. This is illustrated by the prominent QTL (max. LOD= 6.63) for Litter C at the LpACT13G9(4) locus. (Milestone 11).

Trait Linkage Position LOD % variance Locus Kruskal-Wallisgroup (cM) score Explained significance

Herbage C (gC/m2) 4 45 2.21 13.4 LpACA24F2(3) 0.0005

7 38.8 2.36 14.2 LpACT33H5a(7) 0.005

7 43.9 3.88 22.3 LpACA3E2(2) 0.01

7 50.6 3.05 18 LpACA24B11(1) 0.0001

7 54.9 3.56 20.6 LpACA24B11(3) 0.0001

7 62.4 3.1 18.2 LpACT13F9(1) 0.0005

7 74.1 2.44 14.7 LpHCA17C6(2) 0.0005

Litter C (gC/m2) 4 40.2 2.18 13.2 LpACA8B9(2) 0.005

4 54 2.05 12.4 LpACA8B9(4) 0.01

7 43.9 2.58 15.4 LpACA3E2(2) 0.005

7 49.1 6.8 35.7 LpACA24B2(2) 0.0001

7 57.9 6.63 35.4 LpACT13G9(4) 0.0001

7 76.2 3.29 19.6 LpHCA18F11(6) 0.001

Table 3. Comparative positions of QTLs for herbage C content and litter C content on a microsatellite based map (79 genotypes) constructed for the amenity x forage Lolium perenne mapping population. The recorded QTL position is that of the peak LOD score given by interval mapping. The significance level of the Kruskal-Wallis test statistic associated with each of the QTLs is also given. % variance explained = percentage phenotypic variation explained by the QTL.

The results for the mini-swards of 78 progeny and the two parental genotypes of the amenity x forage Lolium perenne mapping family maintained in shallow gradient lysimeters showed in excess of a four-fold range in net litter production in terms of dry matter content (Fig 15A). Data for C and N contents are outstanding, and anticipated to be available by 1 May 2008. (Milestone 11).

SID 5 (Rev. 3/06) Page 22 of 34

05

101520253035

200 400 600 800 1000 1200 1400 More

Num

ber o

f gen

otyp

es

Accumulated litter (kg/ha)

A

R2 = 0.894

8

12

16

20

100 150 200 250 300 350 400N fi

xatio

n (g

N/m

2)

Herbage d.wt (g/m2)

B

Figure 15. (A) Frequency diagrams for net litter accumulated in mini-swards of 80 genotypes belonging to the amenity x forage Lolium perenne mapping family maintained in shallow gradient lysimeters under ambient greenhouse conditons throughout 2007, and (B) Relationship between N2 fixation estimated by cumulative N recovery in herbage cuts 3 and 4 and herbage dry weight across 80 white clover mapping family genotypes grown as mini-swards in shallow gradient lysimeters as described in WP1B (iii). R2

value refer to linear regressions of data.

Objective (ii) Identification of QTLs for traits associated with C sequestration in white clover

BackgroundThere is conflicting evidence regarding the capacity of existing grassland to increase net C sequestration under elevated CO2 without concomitant increases in the net input of other nutrients, particularly N, and/or significant changes in the dynamics of these nutrients in the soil-plant system (e.g. nutrient use efficiencies). However, it is generally recognised that higher nutrient inputs will be required to sustain the increases in net primary productivity (NPP) predicted for many temperate grassland systems under elevated CO2. Greater reliance upon grass-legume mixtures offers a direct method of increasing the N status of pastures and underpinning increases in net primary productivity and below ground productivity. Selecting for enhanced N2 fixation by white clover offers a ‘win-win’ strategy in this context. Hence the work in this project on enhancing C sequestration in mixed grass-legume pastures focussed on identifying QTLs for N2 fixation.

Methods and ResultsThe experimental work was in common with that described in WP1B (iii) with respect to phenotyping the stem nematode resistant white clover mapping family grown as mini-swards in horizontal sand-bed lysimeters. Total N recovered in herbage cuts H3 and H4, during Phase 3 of the experiment, when N acquisition was entirely dependent upon N2 fixation, was used as an estimator of N2 fixation, expressed as gN/m2sward. Whilst these values substantially underestimated the absolute quantities of N fixed during the inter-harvest periods, by omitting the incremental increase in N content of the roots and stolons, and any exudation of N into the rhizosphere, it is likely that variation in the measure reflected in large part the variation in gross N2 fixation within the mapping family. Outline maps were available (resistant and susceptible parent) for the mapping family and interval mapping and Kruskal-Wallis nonparametric single-locus analysis were performed on all mapped and unmapped markers to assess associations between markers and the trait, using MapQTL Version 4.0.

SID 5 (Rev. 3/06) Page 23 of 34

Table 4. QTLs for cumulative N2 fixation, estimated as N recovered in herbage cuts H3+H4 taken from 78 progeny plus two parental genotypes of the ‘stem-nematode resistance’ white clover mapping family genotypes. The recorded QTL positions associated with either the ‘resistant’ or ‘susceptible’ parent are those corresponding with peak LOD scores given by interval mapping. The significance level of the Kruskal-Wallis test statistic associated with each of the QTLs is also given. % variance explained = percentage phenotypic variation explained by the QTL.

Estimated N2 fixation rates on a sward area basis varied four-fold across the mapping family (Fig. 15B) and were highly correlated (r2 = 0.89) with herbage yield. Several QTLs were identified on both parental maps (Table 3), the most prominent of which (LOD= 3.28) was associated with linkage group 3(S-3B) at loci ATS076a and ATS076b. Subject to confirmation of this QTL’s robustness, including determination of G x E effects, the results suggest that this trait may be amenable to marker-assisted selection. (Milestone 12).

Implications and future work (Workpackage 2)Main implications of findingsThe genetic variation demonstrated within the white clover and perennial ryegrass mapping families for traits likely to directly or indirectly affect C deposition into soil, and the identification of putative QTLs for these, suggests that ‘in principle’ it should be possible to pursue a marker-assisted approach to selecting for increased C sequestration in future varieties of forage grasses and legumes. However, whilst the results are encouraging, they can only be regarded as preliminary, not least because the traits assessed were, of necessity, relatively easy to measure and may not be those with the greatest long-term impact on soil C stocks. Furthermore, the agronomic consequences of selecting varieties for C sequestration are unpredictable given the current levels of understanding. The QTLs revealed for nitrogen fixation by white clover will also be of value in marker assisted selection programmes aimed at reducing fertiliser N application to grassland.Future workIn order to provide a sound platform for developing cultivars of forage grasses and legumes with improved capacities for C sequestration, we will confirm the quantitative relationships between grassland soil C levels and species composition, sward age, fertilizer and grazing management, based, where possible, upon the existing literature. We will prioritize traits (a) directly and (b) indirectly affecting C sequestration through variation in (i) C input, (ii) C residence time and (iii) C loss. We will also establish the extent of genetic variation in perennial ryegrass and white clover for the prioritized traits and identify associated QTLs using existing mapping families. This information will be used to develop experimental populations of perennial ryegrass and white clover genotypes with alternative pathways (i.e. traits) conferring enhanced C input to the soil.

3. Improving the stability and resilience of grasslands through the use of multi-species mixtures

Workpackage 3

SID 5 (Rev. 3/06) Page 24 of 34

Parent Linkage Position LOD % variance Locus Kruskal-Wallistype group (cM) score explained significanceresistant 1(R-5C) 23.99 2.51 13.8 PatMaac5 0.0005

3(R-7D) 46.559 1.95 13.1 PacMaat1512(R-8D) 73.824 2.08 39.9 EaacMcca1 0.05

15(R-10E) 41.353 1.87 51.7 PacMaac77(R-13G) 39.789 1.81 23.3 PgaMaga3 0.05

susceptible 11(S-A) 72.652 2.56 58.5 PtaMaac23(S-3B) 41.86 3.28 35.9 ATS076a 0.00053(S-3B) 41.86 3.28 35.9 ATS076b 0.00053(S-3B) 41.948 2.41 13.3 TrAgr1084a 0.013(S-3B) 44.929 2.3 12.8 TrAgr1084b 0.01

14(S-7D) 0 2.48 41 PatMaag414(S-7D) 0 2.48 41 PatMaag4

16(S-12E) 80.506 2.24 35.5 PRS499 0.121(S-15G) 26.992 2.96 46.8 WCBE307 0.121(S-15G) 34.471 1.92 28.7 PatMaac821(S-15G) 43.094 2.37 39.1 PRS510c 0.1

7(S-20?) 45.233 2.37 28.4 PacMaac1 0.05

Objective (i) Effects of multi-species mixtures on agronomic performance

Background Increasing biodiversity in agro-ecosystems is an important policy objective and the relationships between plant diversity, resilience, productivity, resistance to weed invasion and delivery of ecosystem services are key questions of modern ecology. Multi-species mixtures of legumes and grasses offer potential advantages over the perennial ryegrass monocultures and binary white clover/perennial ryegrass mixtures that are usually sown in UK pastures. These potential advantages include greater productivity, more efficient use of environmental resources, a positive impact on soil structure and improved forage quality. Since agriculturally managed grassland constitutes a large proportion of the landscape in many areas of the UK, the level of biodiversity maintained in grassland farming will have a significant impact on biodiversity in the countryside in general. However, the value of increased species diversity in terms of more stable swards, improved resource capture and increased yields with lower inputs has only recently started to be evaluated in an agronomic context, and most research effort to date has been ecologically orientated.We have shown that multi-species mixtures composed of different functional groups (two grasses and two legumes) can be more productive than monocultures in a range of environments. These studies have now been extended to investigate the role of multi-species mixtures of grasses and legumes in the provision of ecosystem services such as reduced nitrate leaching to groundwater, improved soil structure and enhanced forage quality, as well as the production of biomass.

Methods and Results Analysis of biomass productivity in 12 temperate sites (including IGER) participating in a pan-European programme has shown that positive diversity effects persisted over three harvest years at virtually all sites. The experimental design used in this Objective (see below) differs somewhat from that used in the pan-European programme, focusing on the effects of species evenness (relative abundance) on functional outcomes. (Milestone 13).

Objective (ii) Effects of multi-species mixtures on N leaching

Background It appears likely that legume-driven improvements in soil structure and drainage (see Objective (iii)) also directly result in increased leaching of both fixed and applied nitrate in legume monocultures. Indeed, the results of nitrate leaching measurements carried out in Defra commission LS3643 clearly showed that nitrate losses under mixtures of grasses and legumes increased considerably as the proportion of legume tended towards 100%, and were greatly reduced in more complex mixtures (eg. the ‘centroid’, in which sown species proportions were 0.25 for each of four species). Fixed nitrogen from the legume-Rhizobium symbiosis has a key role to play in the development of more sustainable grassland agriculture. However, there is a need to optimise the use of this N and reduce the likelihood of it contributing to diffuse pollution of water courses. The development of complex grass/legume mixtures will potentially overcome the problem of excessive nitrate loss from legumes, as long as the grass component is physiologically capable of carrying out sufficient nitrate uptake and is able to persist over time in sufficient quantities to produce a measurable effect.

Methods and Results Measurements carried out at regular intervals using ceramic cup samplers have shown that nitrate losses under multi-species mixtures of grasses and legumes increased considerably as the proportion of legume tended towards 100%, and were greatly reduced in more complex mixtures. These results suggest that the inclusion of other plant functional groups (eg. grasses) in complex mixtures with legumes might therefore reduce nitrate leaching, as long as the grass component is physiologically capable of carrying out sufficient nitrate uptake and is able to persist over time in sufficient quantities to produce a measurable effect. These questions will be explored further in this Objective. Ceramic cup samplers have been positioned in each plot and will remain in situ as the mixtures develop. They will be sampled at appropriate intervals to provide a statistical comparison of nitrate losses from the eight treatments described below. (Milestone 14).

Objective

SID 5 (Rev. 3/06) Page 25 of 34

(iii) Effects of multi-species mixtures on soil quality

BackgroundThere is some experimental evidence that plant species differ in their visible effects on soil structure and anecdotal evidence has long supported a positive role for legumes in this respect. More detailed investigations of the process of soil structuring have been carried out on white clover and red clover. It has been reported that the changes in soil structuring brought about by white clover resulted in improvements in water percolation rate (i.e. the soil became more freely-drained), and in the extraction by plants of nutrients from the soil. Similar benefits in terms of soil structure have been noted for soil cores under red clover monocultures. Detailed studies of soil structure have shown that soil under actively growing legumes becomes increasingly structured as soil particles become aggregated into clumps, and this process has important impacts on plant/soil interactions. One functional change associated with increased soil aggregation is that the aeration of the soil increases. Previous measurements of oxygen diffusivity (m s-1 x 10-6) obtained in monoculture plots were: red clover (11.9)> white clover (11.1)> perennial ryegrass (7.7)>cocksfoot (6.2). These results support the hypothesis that legumes promote soil structuring processes to a greater extent than grasses do.

Methods and Results The measurements of nitrate leaching in Objective (ii) will be carried out in conjunction with basic assessments of soil quality (soil respiration, infiltration rates, bulk density and aggregate stability) in order to assess the strength of the link between these parameters. (Milestone 15).

To build on these results and allow greater focus on the relationship between soil quality and N leaching under different multi-species mixtures, a field experiment was set up as detailed in Table 5 to 7.

Table 5. Codes and Species used in multi-species mixtures

Code Species Cultivar Commercial Sowing RateL1 Lotus corniculatus (Lc) Leo 10 kg/haL2 Red clover (Tp) AberRuby 10 kg/haG1 Perennial ryegrass (Lp) AberDart 20 kg/haG2 Timothy (Pp) S48 20 kg/ha

Table 6. Treatments: (% commercial sowing rate)

Treatment L1 (Lc) L2 (Tp) G1 (Lp) G2 (Pp)1 91 3 3 32 3 91 3 33 3 3 91 34 3 3 3 915 34 22 22 226 22 34 22 227 22 22 34 228 22 22 22 34

Table 7. Experiment layout

RepPlot No.Treatment

115

128

136

144

153

161

172

187

291

2102

2118

2126

2135

2144

2153

2167

RepPlot No.Treatment

3177

3181

3195

3204

3212

3223

3236

3248

4254

4266

4273

4281

4298

4307

4315

4322

Blocks = 4, Total Plot Number =32, Plot size = 3mx2m, with 50cm paths of an amenity grass separating each plot

SID 5 (Rev. 3/06) Page 26 of 34

Implications and future work (Workpackage 3)Main implications of findingsThe relationship between biodiversity and productivity is currently a central issue in plant ecology and has been the subject of recent investigation using experimental plant communities with different levels of species richness (number) (eg. the multi-site BIODEPTH project (Hector et al., 1999, Science 286, 1123-1127)). Most research in this area has used simplified, small scale, systems, focusing on the effects of species and functional group identity and species richness on community function (reviewed by Hooper et al., 2005, Ecological Monographs 75, 3-35). However, it has become clear that diversity effects in plant communities are also produced by inter-specific interactions, the strength of which depends on the relative abundance (evenness) of the species involved (Kirwan et al., 2007, Journal of Ecology 95, 530-539). Many studies have described the changes in relative abundance of species that occur as a response to competition in plant communities but few have used robust experimental designs to vary evenness systematically in multi-species mixtures, and until recently analytical procedures have not incorporated the joint effects of evenness and richness. The novel experimental design (‘simplex’) used by Kirwan et al. (2007) directly addressed these issues in an agronomic context. This experiment was established under the auspices of an EU COST Programme (Action 852) at a large number of sites using carefully defined levels of species richness and evenness. All sites used mixtures containing four species: two legumes and two grasses. Two IGER sites (Aberystwyth and Bronydd Mawr) participated in the experiment using red clover, white clover, perennial ryegrass and cocksfoot. There was clear evidence of transgressive overyielding in both sites (i.e. multispecies grass/legume mixtures had significant yield advantages over the same species growing in monoculture). There was also a negative and persistent diversity effect on the weed content of swards, with much lower weed contamination of mixtures than monocultures of any species. Future workWhilst biomass production is obviously of key importance, other properties of mixed swards are also of agronomic and environmental interest and it is clear that there is scope for further progress to be made in the design of multifunctional grass/legume mixtures based on more precise information concerning the effects on the environment of specific physiological traits of the individual components. There are three main areas in which the attributes of mixed swards can make a contribution to reducing the environmental footprint of grassland agriculture, whilst simultaneously delivering benefits in terms of the nutrition and health of grazing animals: (i) increased biomass production and fewer weeds (ii) improved soil quality and reduced nitrate leaching; (iii) improved forage quality to improve the efficiency of protein utilisation in the animal rumen. This Workpackage uses a modified version of the simplex design employed by Kirwan et al. (2007) in order to analyse the impact of multispecies grass/legume mixtures on these important aspects of ecosystem functioning.

4. Enhancing adaptation to climate change through improved water use efficiency and drought tolerance

Workpackage 4

Objective (i) Quantify drought tolerance and WUE of advanced hybrids betyween white clover and

Trifolium ambiguum.

BackgroundIn white clover, the potential of introgressing the rhizomatous trait from the closely related drought tolerant species Trifolium ambiguum (Caucasian clover) to improve tolerance of drought has been demonstrated. Preliminary studies on BC2 hybrids has shown that there is improved tolerance of drought compared with the white clover parent however further selection and backcrossing has been carried out to increase fertility (seedset) to a level appropriate for commercial development and prior to entry into NL trials. Advanced backcross 3 hybrids between white clover and Caucasian clover have now been developed which are white clover like in appearance but which combine an essentially stoloniferous growth habit but with a proportion of their dry matter as rhizomes with no detrimental effect on dry matter production or N fixation. Whilst the drought tolerance of these hybrids in comparison with white clover is important, water use efficiency (amount of water used per unit of dry matter production) is also an important trait for climate change scenarios where increased summer

SID 5 (Rev. 3/06) Page 27 of 34

drought is predicted. Similarly, identification of the traits underpinning these differences and the extent of variation in different backcross generations are important.

Methods and ResultsExperiments in 1m deep polythene lined soil pipes and in deep soil bins (1m x1m x 1m) were carried out to determine the drought tolerance of advanced hybrids in comparison with the white clover parent and quantify differences in their root development (Fig. 16). Differences in root distribution between Caucasian clover and the white clover parent were clear yet there was no significant difference between white clover and the BC1 and BC2 hybrids despite these hybrids being able to maintain a higher leaf relative water content and leaf water potential than the white clover parent and maintain a higher rate of stolon growth under drought (using Carlson scale).

Table 6. Leaf water potential (MPa) of T. repens, T.ambiguum, BC1 and BC2 hybrids after 21 and 35 days at three levels of soil moisture deficit. C- control treatment, M- moderate moisture stress, S-severe moisture stress. Data is expressed as the mean of four genotypes + s.e.m.___________________________________________________________________Moisture Days of Species/hybridlevel experiment T. repens T. ambiguum BC1 BC2 ___________________________________________________________________C 21 -0.40+0.042 -0.70+0.100 -0.63+0.042 -0.38+0.039

35 -0.59+0.081 -0.75+0.072 -0.43+0.081 -0.46+0.046M 21 -0.78+0.191 -0.87+0.080 -1.05+0.210 -0.62+0.111

35 -1.27+0.160 -1.09+0.072 -1.23+0.292 -0.93+0.152S 21 -1.37+0.134 -1.14+0.039 -1.20+0.075 -1.41+0.121

35 -3.30+0.465 -1.09+0.046 -2.54+0.083 -2.25+0.041

0 5 10 15 20 25 30 35 40 45 50

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

Dep

th (m

)

Root weight (g)

T.ambiguumT. repensBC1BC2

Figure 16. Root weight of 10cm segments of the root profile of T. repens, T ,ambiguum and BC1 and BC2 hybrids. Vertical bars represent the s.e. of the mean. n= 12.

Small scale studies of the BC3 hybrids show that these are essentially white clover like in appearance yet retain the improved drought tolerance of the Caucasian clover, which may be due to differences in stomatal density or better regulation of leaf water content. This material will form the basis for future commercial varieties.

An experiment was also carried out in pots using four clonal plants of a range of backcross hybrids to compare the water use efficiency (WUE = g DM/ kg water uptake) of these hybrids in comparison with

SID 5 (Rev. 3/06) Page 28 of 34

white clover and Caucasian clover. Using techniques developed at IGER for measuring WUE on large numbers of plants we have measured water usage per plant per day and dry matter accumulation in root and shoot. WUE of the white clover varieties Menna and Alice was 470ml/g DM and 455ml/g DM respectively whilst the backcrosses ranged from 347ml/g DM to 507ml/g DM indicating significant variation for this trait within the hybrids. WUE of the Caucasian clover was higher (618ml/g DM) however this was attributed to the lower DM production and slower growth rate inherent to this species rather than water usage. Future work will determine whether carbon isotope discrimination (used successfully in other species to measure WUE) can be used to identify material. The outcome of this work will be identification of key traits associated with improved drought tolerance and greater WUE which combined with marker based approaches will lead to the development of genetic material which can be integrated into germplasm improvement programmes. Further development of these hybrids will result in submission for NL trials and will be facilitated by the use of a molecular marker diagnostic for the rhizomatous trait. (Milestone 16).

Objective(ii) To assess the extent to which ryegrass plants growing over 1 year in 1m deep polythene-

lined pipes and carrying fescue genes from chromosome 3 have enhanced rooting characters in both drought stressed and irrigated growth conditions.

BackgroundFescues are characterized as having larger and deeper roots than ryegrasses which contributes to their greater water-use-efficiency and drought resistance. In previous studies, genes for drought resistance were transferred from chromosome 3 of tall fescue (Festuca arundinacea) and of F. glaucescens into Italian ryegrass where they contributed to highly significant increased drought resistance and capability of survival and recovery following 3 summer months with no irrigation.

Methods and ResultsA drought experiment was conducted on the introgression ryegrass lines containing F. arundinacea and F. glaucescens genes for drought resistance and on control varieties of ryegrass and fescue parents plants in pipes to measure changes in root development brought on in response to the drought stress. To support this study a series of recombinant ryegrass plants were used, each containing different meadow fescue (F. pratensis) DNA sequences found on chromosome 3 to assist with targeting the genes for rooting traits.

Replicate clones of chromosome substitution lines, the recombination series for sub-line chromosome 3 and the introgression lines were established successfully in 1m deep polythene-lined pipes. Measurements for root growth, general productivity and performance, and response to drought were carried out between April and September 2007. Five cuts were taken, in the weeks starting April 16th, June 4th, July 2nd, August 6th, and September 17th. Above ground growth was assessed as tiller counts and as fresh and dry matter. Floral development was monitored throughout the spring and early summer. Non-destructive root measurements were also carried out during the first and last sampling weeks in April and September. Maximum depth, root scores for each 10cm horizon, the proportion of new young (white) roots for each 10cm horizon, a lateral root score and a root hair score were recorded. Some additional trait values were derived from the data. Root system size was the sum of the scores for all the horizons and root profile was fitted by regression through root scores in each horizon. By the autumn, there were differences in root growth within the full set of genotypes for maximum root depth (P<0.001), root system size (P<0.001), the frequency of lateral roots (P<0.001), of root hairs (P<0.001) and of new root growth (P<0.001).

The drought commenced in half the pipes on July 2nd and finished on August 20th when the droughted pipes were rewatered. Measurements were carried out in mid and late July, and throughout August. Wilting was scored, leaf water potential (dawn and mid-day) measured, and samples taken for relative water content and osmotic potential (both direct and rehydrated). After the droughted pipes had been rewatered, regrowth was recorded as both tiller survival and leaf extension. Within the sub-line 3 recombination series some contrasting root responses to drought were identified (Fig. 17). As example Fig.1 illustrates reciprocal recombinants, plant 922 (where circa 90% of chromosome 3 is meadow fescue), and 924 (where the equivalent chromosome segment is replaced by perennial ryegrass DNA sequences) where 922 increases proportionately root growth and root depth during drought stress in

SID 5 (Rev. 3/06) Page 29 of 34

contrast to plant 922 which produced more roots close to the soil surface under the same drought conditions. (Milestone 17).

ROOT PROFILE

806 810 820 822 831 843 848 920 921 922 923 924 925 926 927 929 930 931 933

LSD

WATERED

DROUGHTED

922 924

Figure 17. Effect of drought on root profile for the sub-line 3 recombination series.

Objective (iii) Development of selection criteria within white clover for improved soil quality

BackgroundWhite clover roots have been shown to improve soil quality and can change the soils hydraulic properties by altering its water repellance, which can influence water use efficiency. We are translating new knowledge of the beneficial impact of white clover on soil quality, aeration and oxygen diffusivity into selection criteria for the development of new varieties.

Methods and ResultsIn white clover, a relationship between stolon traits and root architecture has already been established. To develop selection criteria that will enable the development of advanced germplasm with improved root traits we have used a white clover mapping family developed to investigate effect of differences in stolon morphology on root characteristics. Comparisons of the parents (T1 and T2) of this white clover mapping family grown in 1m soil pipes show significant differences in the density of roots in the root profile between the two parents (Fig. 18). This mapping family will be used to study the effect of root characteristics on soil quality and water movement and offers the potential to identify QTL associated with soil quality traits. (Milestone 18).

SID 5 (Rev. 3/06) Page 30 of 34

Figure 18. Root density score of T1 and T2 (white clover stolon mapping family parents at 10cm segments of the root profile. Vertical bars represent the s.e. of the mean. n=8

Objective(iv) Assess the extent to which ryegrass plants growing over 1 year in 1m deep polythene-

lined pipes and carrying fescue genes from chromosome 3 have enhanced rooting characters

BackgroundA previous Defra programme LS3647 located genes for root production in a highly diverse perennial ryegrass population produced from parental genotypes with diverse water soluble carbohydrate (WSC) expression. Here using the same population root growth was measured in a replicated experiment in polythene-lined 1m deep pipes during spring and autumn. The project design will allow direct comparisons to be made with Objective 4(ii) and the impacts in ryegrass of new variation on root development derived from the presence of fescue genes to be determined.

Methods and ResultsWork is ongoing with the WSC F2 mapping family grown in deep pipes. Root measurements were taken previously in 2006 and in the current programme in 2007. Preliminary QTL analysis in the previous Defra programme had already corroborated data for crude scores of root amount in pots. Calculation of derived parameters has been further refined following work with Festulolium material as in 4(ii) above. QTL analysis will be finalised when a fully replicated data set has been collected.

The WSC F2 mapping family has also been grown in small 8.5cm sand columns in a fully replicated experiment. Shoots and roots were sampled after 14 weeks growth. Tiller number, shoot dry matter, root dry matter and root/shoot ratio will be measured. There appeared to be considerable variation in plant-to-plant nutrient acquisition during the experiment. Therefore, all sampled material is being freeze-dried to permit the future analysis of various metabolite fractions in the tissues. Trait variation will be analysed and QTL analysis carried out. In future the sand column system will be further developed to include use of wax layers developed for use in rice to measure rot penetration through hard compacted soils. (Milestone 19).

Implications and future work (Workpackage 4)Implications of main findings

SID 5 (Rev. 3/06) Page 31 of 34

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

0-10 10-20 20-30 30-40 40-50 50-60 60-70 70-80 80-90

Segments

Roo

t den

sity

sco

re

TIT2

We have shown that backcross hybrids between white clover and Trifolium ambiguum, selected on the basis of incorporating the rhizomatous trait of and that are essentially white clover like in appearance are more drought tolerant than the white clover parent. We have also shown differences in water use efficiency between the parental material and the hybrids indicating the potential of using this material to select varieties that are more drought tolerant but also more efficient in their use of water. Differences in the root architecture of the parents of a white clover mapping family, developed to study aspects of stolon development, have been identified. This confirms the relationship between stolon traits and root growth and provides a mapping family that offers the potential to study the impact of root architecture on water movement and the potential to identify QTL associated with root traits and soil quality to be quantified.Detailed scores were made of root growth in pipes comprising a recombination series for F. pratensis chromosome 3 where fescue chromosome segments of different size and location were introgressed into L. perenne chromosome 3 in an otherwise undisturbed L. perenne genome. Root and foliar growth measurements were taken from clones of the same genotypes under irrigated and droughted conditions to assess the drought effects. Re-growth was also scored subsequent to completion of the drought test. Between genotypes significant differences were found in root depth, root size, lateral roots, root hairs and new root growth. Differences were recorded in root growth between genotypes grown under irrigated and under droughted conditions. Genotypes that increased rooting depths under droughted conditions were identified which is likely to assist their subsequent survival. Contrasts between genotypes in root growth response under drought conditions have assisted the location of those fescue-derived genes responsibleFuture workSelection lines of the hybrids will be developed into commercial varieties and we will determine whether carbon isotope discrimination can be used to quantify WUE. We will quantify differences in the root architecture of the white clover stolon mapping family and identify QTL associated with root traits. WE will also use this material to quantify the impact of root traits on soil quality in monocultures and mixed swards and relationship between root traits and soil quality. This will lead to the development selection criteria for root traits that can be integrated into the white clover breeding programme.In previous research chromosome 3 of fescue species has been demonstrated to be an excellent source of novel genes for ryegrass for drought resistance traits. One explanation was that genes for increased root growth reside within this chromosome and assist when water supply is limited to better access water resources lying deep in the soil. The current research demonstrated contrasts in root growth under drought stress between genotypes with alternative ryegrass and fescue gene variants and therefore gave some support to this explanation. More detailed investigations into water-use-efficiency to be achieved both in plots and by spaced-plants including these ryegrass genotypes and others with fescue genes for drought resistance will be undertaken in forthcoming DefraLINK project LK0688. In this project the genes for drought resistance will also be transferred from L. multiflorum into L. perenne where they can be used more effectively in developing new drought resistant ryegrass cultivars that are suitable for commercial exploitation in the UK.

Actions arising from this workThe knowledge, germplasm and molecular markers developed in this work will feed through into IBERS variety development. From April 2008 work directly leading to the development of new varieties will be funded through the Sustainable Livestock Production LINK Programme. The traits and approaches described in this report do not currently form part of this Programme but following further development they will be utilised in variety development through the same ‘pipeline’. Variety finishing and commercialisation is carried out by Germinal Holdings Ltd and Aberystwyth University will be able to obtain Plant Breeder’s Rights on new varietiesThere is a strong route to market which has been successful in introducing innovative varieties to a large number of UK livestock farmers. This is supported by extensive technology transfer carried out through a number of routes including the IBERS Grassland Development Centre. Technology transfer is also supported by the meat and milk levy boards and the BGS.The outcomes from this work will also be disseminated directly to farmers and policy makers in the form of guidelines, talks, demonstrations at shows and events, articles in the farming press and contributions to, for example, BGS meetings.

SID 5 (Rev. 3/06) Page 32 of 34

References to published material9. 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.Refereed journalsPublished/ Submitted / Approved / In Press

Abberton, M.T., MacDuff, J., Marshall, A.H. and Humphreys, M.W. The genetic improvement of forage grasses and legumes to enhance adaptation of climate change. FAO publication (in press)

Abberton, M.T., MacDuff, J., Marshall, A.H. and Humphreys, M.W. The genetic improvement of forage grasses and legumes to reduce greenhouse gas emissions. FAO publication (in press)

Abberton, M.T., Marshall, A.H.,Humphreys, M.W. MacDuff, J., Collins R.P. and Marley, C. Genetic improvement of forage species to reduce the environmental impact of temperate livestock grazing systems. Advances in Agronomy (98) 311-355 (in press).

Humphreys, M. W., Marshall A.H., Yadav R., Abberton M.T. (2008). Ameliorating climate change impacts by improving the drought resistance of UK grasslands. Journal of Agricultural Science (submitted)

Lüscher, A., Finn, J.A., Connolly, J., Sebastià, M.T., Collins, R.P., Fothergill, M., .Porqueddu, C., Brophy, C., Huguenin-Elie, O., Kirwan L., Nyfeler, D., Helgadóttir, Á. Benefits of sward diversity for agricultural grasslands. Biodiversity (in press).

Marshall, A. H. , Bryant, D. N. , Latypova, G. A. , Olyott, P. , Morris, P. , Robbins, M. P. (2008) A high throughput method for the quantification of proanthocyanidins in forage crops and its application in assessing variation in condensed tannin content in breeding programmes for Lotus corniculatus and L. uliginosus Journal of Agricultural and Food Chemistry 56, 974-981.

Marshall, A. H. , Michaelson-Yeates, T. P. T. , Abberton, M. T. Introgression of reproductive traits from Trifolium nigrescens increases the seed yield of white clover (Trifolium repens L.) Plant Breeding (accepted)

Turner, L. B. , Cairns, A. J. , Armstead, I. P. , Thomas, H. , Humphreys, M. W. , Humphreys, M. O. (2008). Does fructan have a functional role in physiological traits? Elucidation by QTL mapping. New Phytologist (in press)

Conference papersCollins, R. P., Fothergill,M., Rees,E., Kirwan, L., Brophy, C. and Connolly, J. Site effects on species productivity and dynamics: results from COST 852 in Wales. Grassland Science in Europe (in press).

Fothergill, M. , Abberton, M. T. , Collins, R. P. , Michaelson-Yeates, T. P. T. , Marshall, A. H. , Rees, M. E. (2007) Breeding forage legumes for a new era Proceedings of final meeting of COST Action 852, Raumberg-Gumpstein, Austria, 30 August - 3 September 2006

Hawkins, SL., Turner, LB., Humphreys, MW. (2007). “Selecting Genes in Lolium x Festuca Hybrids for Root Growth to Improve Soil Hydrology.” Eucarpia Symposium On Improvement Of Fodder Crops and Amenity Grasses, Copenhagen (Denmark), August 19-23 in Copenhagen.

Helgadóttir, Á., Connolly, J., Collins, R.P., Fothergill, M., Kreuzer, M., Lüscher, A., Porqueddu, C., Sebastià, M.T., Wachendorf, M., Brophy, C., Finn, J., Kirwan, L. and Nyfeler, D. The benefits of sward diversity for cultivated grasslands.Grassland. Science in Europe (in press).

Humphreys, M. W., Binley, A., Clark, L. J., Hawkins, S. L., King, I. P., Macleod, C. J. A. et al. “Grass roots to improve the management of soil-water” 'High Value Grassland: providing biodiversity, a clean environment and premium products'. A joint BGS/BSAS/BES Conference, University of Keele, Staffordshire, 17-19 April 2007.

Humphreys, M. W., Harper, J. A. (2008). Festulolium loliaceum, an understudied natural UK grass hybrid species that may provide benefits to UK grasslands withstanding the onsets of climate change. Crop Wild Relative (6) January 2008,

SID 5 (Rev. 3/06) Page 33 of 34

Humphreys, M. W., Turner, L. B., Binley, A., Hawkins, S. L., Price, Z., Skøt, L., Macleod, C. J. A.  , Whalley, W. R., Papadopoulos, A. & Haygarth, P. M. (2008). Selecting Genes in Lolium x Festuca Hybrids for Root Growth to Improve Soil Hydrology. Oral presentation by M. W. Humphreys at Molecular Mapping & Marker Assisted Selection in Plants, February 3-6 2008, Vienna, Austria.

Macleod, C. J. A. , Binley, A. , Clark, L. J. , Hawkins, S. L. , Humphreys, M. W. , King, I.P. et al. (2007). Genetically modified hydrographs: what can grass genetics do for temperate catchment hydrology? European Geosciences Union, General Assembly 2007, Vienna, Austria, 16-20 April 2007

Marshall, A. H. , Abberton, M. T. , McCalman, H. M. (2007) Providing forage varieties and seeds for the organic livestock sector Online at: \\Wpnetapp1a\protrak\PUBLICATIONS\EUCARPIA2006\proceedings2006.pdf 'Breeding and seed production for conventional and organic agriculture'. Proceedings XXVI EUCARPIA Fodder Crops and Amenity Grasses Section and XVI Medicago spp. Group joint meeting, Perugia, Italy, 3-7 September 2006   Rosellini, D. , Veronesi, F., eds. 38-46.

Marshall, A. H. , Abberton, M. T. , Thorogood, D. , Armstead, I. P. , Michaelson-Yeates, T. P. T. (2007) Strategies to combine improved reproductive and agronomic traits in forage plant breeding programmes 6th International Herbage Seed Conference, Norway, 17-23 June 2007   22-27

Marshall, A. H., Collins, R. P., Fothergill, M., Abberton, M. T. (2007) Enhancing delivery of ecosystem services through forage legume breeding 'High Value Grassland: providing biodiversity, a clean environment and premium products': Proceedings BGS/BES/BSAS Conference, Keele University, Staffs, 17-19 April 2007. British Grassland Society Occasional Symposium, 38 Hopkins, J. J. , Duncan, A. J. , McCracken, D. I. , Peel, S. , Tallowin, J. R. B., eds. 273-276.

Marshall, A. H. , Collins, R. P. , Fothergill, M. , Abberton, M. T. (2007) Improving forage and seed yield in temperate forage legumes Zbornik radova - Institut za ratarstvo i povrtarstvo (Serbia)   Symposium on Forage Crops XI, Novisad, Serbia, 27 May - 4 June 2007   Kobiljski, B., eds. 27-33.

Rees, M. E. , Fothergill, M. , Marshall, A. H. (2007) Effect of white clover companion on the establishment of contrasting varieties of Lotus corniculatus in low fertility conditions Proceedings of final meeting of COST Action 852, Raumberg-Gumpstein, Austria, 30 August - 3 September 2006

Skøt L., Thorogood D., Armstead I. P., Humphreys J., Sanderson R., Thomas I., Gallagher J., Turner L. B., Humphreys M. W., & Humphreys M. O. (2008). Association and introgression mapping of quantitative traits in Lolium perenne: Flowering time, forage quality and abiotic stress tolerance. Oral presentation by L. Skøt at the Plant and Animal Genomics Conference XVI, January 12-16, 2008. San Diego, USA.

Yadav R S, Roderick H W, MacDuff J, Humphreys M W: Genetic improvement of traits conferring enhanced economic and environmental sustainability to grassland agriculture. XXVIIth EUCARPIA SYMPOSIUM ON IMPROVEMENT OF FODDER CROPS AND AMENITY GRASSES, Copenhagen, August 19-23 (Denmark), 2007

A wide range of information gained from these studies has also been included in articles to the farming press and press releases. We have also actively disseminated information at numerous agricultural shows, events and to visitors to the Institute.

SID 5 (Rev. 3/06) Page 34 of 34