Public Privat Partnership (PPP) for Pre-breeding. PPP for pre-breeding in perennial ryegrass (Lolium perenne L.)

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1 Call: Project title: Public Privat Partnership (PPP) for Pre-breeding PPP for pre-breeding in perennial ryegrass (Lolium perenne L.) Introduction In Northern Europe the expected climate changes will result in new growth conditions for forage production due to an extended (1-3 months) growth season combined with milder and rainier autumns and winters 1. An example of the expected changes in the length of the growth season for Norway is shown in Fig. 1. Similar changes are expected for regions of the Nordic countries at comparable latitudes, while regions further south (Denmark and Southern Sweden) might experience increasing drought problems during the growth season. Fig. 1. (a) Length of growth season (days) in Norway, average for the reference period (b) Predicted increases (days) in the length of the growth season for the period Perennial plants like forage grasses and legumes show local adaptation to temperature and photoperiod. Important physiological processes governing plant phenology (vernalization, cold acclimation, heading and flowering) are determined by temperature and photoperiod and their interactions. This in turn determines winter survival and seasonal yield distribution. The seasonal photoperiod does not change with the climate changes that cause the temperature to rise. This raises a need for new ideotypes with a different set of physiological traits than the present cultivars. An increased risk of mild periods during winter causing plants to deacclimate (deharden) too early, increased risk of frost injury in spring in some locations, and increased frost exposure during winter in others given that the current cultivars are used are some of the 1 Hansen-Bauer I (ed.) Klima i Norge Bakgrunnsmateriale til NOU Klimatilpassing. Foreløpig utgave juni endelig_lavoppl.pdf 2 Skaugen TE, Tveito OE. Growing-season and degree-day scenario in Norway for Climate Research 26: ,

2 expected consequences. A pertinent question is thus whether the available germplasm and active breeding populations are sufficiently broad, or exotic materials should be identified, introgressed by crossing and recombined, to serve as new genetic resources for development of cultivars for the future climate at higher latitudes. We should remember that all major herbage plant species have been introduced to higher latitudes in the recent few hundred years, likely from a few introductions, and they probably harbour a restricted amount of genetic variation due to founder and bottleneck effects. A PPP pre-breeding programme in perennial ryegrass Perennial ryegrass (Lolium perenne L.), which is the main forage grass species in Denmark and south-wards in Europe, is at present at the border of its adaptation when grown north of a line Oslo to Helsinki. Perennial ryegrass has superior feed quality and productivity under frequent cutting regimes, and is expected to expand further north due to milder winters with shorter periods of snow cover. The main weaknesses are susceptibility to low-temperature pathogens, inadequate growth cessation in the autumn to allow for sufficient cold hardening and winter survival, with low persistency as the result. In order to prepare for the predicted climate changes a PPP for pre-breeding programme aimed at selecting plant materials of perennial ryegrass for the development of cultivars with a suitable adaptation to future climates in the Nordic countries is outlined in the following. Breeding of perennial grasses is a long-term activity which require many years from the starting exploitation of germplasm until the release of improved cultivars. A successful breeding programme relies very much on proper pre-breeding activities, and pre-breeding is also a continuous, long-term activity. We present a detailed programme for the first phase ( to ) of a PPP pre-breeding programme for perennial ryegrass and outline plans for a second phase ( ). The project is organized in 6 work packages (WP) in the first phase ( ), with a planned extension from 2013 with 4-5 WPs. A flow-chart of the programme is presented in Fig. 2. Links to other networks and projects Several of the partners, i.e. UMB, Univ of Aarhus, Agric Univ of Iceland (AUI), DLF and Graminor, are members of the Nordforsk network NOFOCGRAN (Nordic Forage Crops Genetic Resource Adaptation Network ( ). The aims of the network is The main objective is to develop a strong network of researchers and students involved in the development of knowledge, methods and germplasm as the basis for future development of cultivars of perennial forage grasses and legumes for the Nordic region. The network is coordinated by Prof. OA Rognli. NOFOCGRAN and the present PPP for pre-breeding project will benefit mutually from each other and further consolidate the collaboration between the Nordic community involved in genetics and breeding of forage crops. Also knowledge and technology developed in the project VARCLIM - Understanding the genetic and physiological basis for adaptation of Norwegian perennial forage crops to future climates ( ), funded by the Research Council of Norway (NFR), with UMB and Graminor among the partners, will be useful for the present PPP pre-breeding project. 2

3 Project outline : Project leader: Prof. Odd Arne Rognli, Norwegian University of Life Sciences (UMB), Ås, Norway Project partners (contact persons): DLF (Niels Christian Nielsen), Lantmännen (Christer Persson), Graminor (Petter Marum), Agricultural University of Iceland (Aslaug Helgadottir), Jõgeva Plant Breeding Institute (Rene Aavola), Boreal (Miko Isolathi), UMB (Odd Arne Rognli), and University of Aarhus (Torben Asp). Project management: The project will be managed by the project group composed of the contact persons representing the project partners. In this first phase of the programme it is very important to meet regularly for detailed planning of the work and for building confidence and trust between the partners. Therefore two project meetings will be organized per year, at different partner locations, during this first phase of the project. The contact persons listed above will be responsible for leading the different work packages (WPs) described below and for the partner contributions to the WPs. Project budget: The total cost for the project is DKK 3,143,700. The companies and NMR contribute equal parts of DKK 1,571,850. Detailed budget figures are presented in Tables 2-6. WP1: Locating and requesting Lolium perenne L. accessions and cultivars Responsible: Graminor Participating: Graminor and DLF Timeframe: 2012 In order to get a good screening of genetic variation in Lolium perenne, a substantial number of accessions are needed. The aim in this project is to acquire a total of 400 accessions. These accessions will be used in WP2 (Seed multiplication), WP4 (Phenotyping), WP5 (Broad breeding population), and WP6 (Genetic diversity analyses). We will need at least 500 viable seeds of each accession. In addition we aim at getting seed of up to 20 commercial cultivars with very good winter hardiness. The cultivars will be tested in WP3. A large number of accessions of Lolium perenne L. are stored in Europe, North America, and Japan (Table 1.). The EURISCO database gives information on 9264 accessions stored in European gene banks. These accessions originate from a total number of 57 countries. The largest number of accessions originates from United Kingdom, Germany, Ireland and the Netherlands. The European Cooperative Programme for Plant Genetic Resources (ECPGR) has a crop database for Lolium. The ECPGR database holds a bit more information about the accessions than the EURISCO database which only holds passport data. Accessions stored in North America can be located in the GRIN database and material stored in Japan in the NIAS database. 3

4 It is important to assemble a different gene-pool with different genetic variation compared to what the European breeders are utilizing today. Thus accessions that originate from northern latitudes and also from more extreme climates e.g. Turkey and Afghanistan should be selected. If plant materials from Western European are included, the number of accessions should be limited. Landraces should be selected first if found, followed by cultivars and wild/semi-wild populations. Table 1. Number of accessions of Lolium perenne L. listed in different gene bank databases Gene bank Number of accessions EURISCO (European genebanks) 9264 Nordgen (included in EURISCO) 203 Vavilov (included in EURISCO) 431 GRIN (North America) 641 NIAS (Japan) 172 These databases will be used to locate and select the 400 accessions for this project. Nordgen has good contacts with other gene banks. It will therefore be most efficient for the project to procure services from Nordgen personnel in requesting accessions from other gene banks and to handle the MTAs that are required. WP2: Multiplication of gene bank accessions for further testing Responsible: DLF Participating: DLF Timeframe: The number of seeds received per accession will be limited. The primary task is to grow plants for the seed multiplications. The second task is to do phenotyping (WP4) of the accessions and/or a broad base population at the same time as the seed multiplication is done if enough plants can be established from the seed lots received. DLF will receive the seed samples from the gene banks and will establish plants both for the seed multiplications at DLF and for distribution to Graminor, Lantmännen, and Boreal WP4 (phenotyping) and WP5 (establishment of a broad breeding population) activities, respectively. The establishment of all plants at one location is done in order to minimize the risk of reducing the germination of seed samples by splitting and distributing them to different locations. The multiplications will be made in 16 m 2 plots planted with plants to represent the accession. The planting will take place in June and for isolation a 4 m wide border of rye/wheat mixture will be sown around each plot. Observations of establishment will be done in autumn 2012 and on survival in spring Other traits, e.g. disease resistance, will be scored if judged necessary. The multiplications will be harvested with a Wintersteiger combine fitted with a compressor so that the combine can be cleaned between plots. The cleaning will take place using a Petkus cleaning machine. The amount of cross contamination and admixture is expected to be 4

5 negligible. A seed sample of 500 to 800 g can be expected from each plot. The seed samples will be cleaned and ready for the next steps in the project in December 2013/January WP3: Testing the potential winter hardiness of existing perennial ryegrass cultivars Responsible: Agricultural University of Iceland (AUI) Participating: AUI, Lantmännen, Graminor, Boreal, and Jõgeva. Timeframe: The aim of this work package is to test existing cultivars of exotic origin in order to identify possible sources of germplasm for improving winter hardiness. Under the auspices of the NOFOCGRAN network, trials with Nordic cultivars of ryegrass and Festulolium have already been established in Norway, Iceland, Finland and Denmark and will likely be established in Sweden in spring Results from these trials will be made available for the present project. Hence, the planned trial here will concentrate on exotic materials. The 20 winter-hardy cultivars collected in WP1 during the winter 2012 will be established in spring 2012 or early summer 2012 at the following locations: Korpa Experimental Station in Iceland, Lännäs Experimental Station in Sweden, Bjørke Experimental Station in Norway, Jõgeva Plant Breeding Institute in Estonia, and Jokioinen Experimental Station in Finland. Seed will be sown in plots of standard size for cultivar trials with three replicates at each location. The plot cover will be recorded in autumn of the sowing year before winter sets in. In subsequent years ( ) the plots will be scored for winter survival and type of winter damage in spring and the dry matter yields obtained during the growing season will be measured. Work for the next phase of the project (2014-?) Observations of the field trials will continue in 2014 and Surviving plants at the different locations will be dug from the fields, genotyped with suitable trait-associated markers (publicly available or developed during the first phase of this project in WP6) and intercrossed to provide regional-specific populations for further cultivar development. Task 4 (WP4): Phenotyping of gene bank accessions Responsible: Lantmännen Participating: Lantmännen, Graminor, DLF and Boreal Timeframe: The 400 accessions of perennial ryegrass will be phenotyped for several traits. 5

6 The important traits are: Winter survival Spring growth Growth habit Heading time Plant height Disease attack, especially crown rust The responsibility for phenotyping the traits will be divided among the participants. Crown rust attack is most frequent in Denmark and winter damage is likely to be most severe in Norway. But the traits can be observed also by the other participants if there are differences in the trait at their location. The observations will be made on 20 single plants per accession at each location in The observation plots in Sweden, Norway and Finland will be kept for two years to allow for observations of winter damage after two winters. Based on the results from these trials subbreeding populations selected for different traits, e.g. winter survival will be established (see Fig.2). Work for the next phase of the project (2014-?) Observation of winter damage will continue in When seeds of the accessions have been multiplied it will be possible to sow plots and make observations on dense stands. This is important for observations of snow mould injuries. Task 5 (WP5): Establishing a broad breeding population (Base broadening) Responsible: Graminor Participating partners: Graminor and DLF. Timeframe: One of the goals of this pre-breeding project is to increase the variation in our breeding material. This WP will create a broad breeding population in diploid perennial ryegrass. This breeding population will be the basis for the pre-breeding work with a time frame of years or more. The breeding population will be based on the 400 accessions mentioned in WP1. From each accession 10 plants will be used in the crossing block. In order to reduce the effect of selection for differences in response to photoperiod and other climatic factors at this stage, two crossing blocks, one in Denmark and one in Norway, each with a total of 4000 plants will be established. The plants will be completely randomized within each crossing block. The plants will be raised in a greenhouse at DLF together with the plants for WP2 and WP4 and transported to Norway. At each location seeds from one half of the crossing block will be harvested early and the other half will be harvested late. The first generation breeding population will be established by mixing equal amounts of seeds from these four different seed lots. 6

7 In addition one seed head will be harvested from each plant and used to establish one plant per seed head for production of the second generation (SSD-single seed descent). This will be done to in order to reduce selection in the material at this stage. The second generation will be produced in order to increase the recombination of genes or gene blocks. Work for the next phase of the project (2014-?) After the creation of this breeding population, it can be used in a number of ways. This will be done in WP8 in the next phase of the pre-breeding programme. a) The broad breeding population will be sown in the field in different climatic zones in the Nordic countries including Estonia. The populations will be subjected to selection pressure by harvesting for 1-2 years using a harvest regime typical for the region. In the second or third year we will harvest seed at the different locations. This process will be repeated by establishing a new trial at the same location with seed from that location. Over time the populations will adapt to the growing conditions at the sites and also adapt to the gradual changes in climate. It is predicted that the growing season will be longer, new diseases like rust will be more important and the winters will be more variable. A similar Norwegian project has already been running for 8 years in timothy, meadow fescue and red clover 3 The genetic changes in these populations will be studied using molecular markers. Knowledge and technology developed in the project VARCLIM where shifts in allele or haplotype frequencies as a result of local adaptation is being studied will be used to design studies in the new populations. b) The broad breeding population can also be used in an alternative way, to create a population with wide adaptation. In this alternative we will start in the same way as in a), however, the seed for establishing the next trial at each location will consist of a balanced mixture of seeds from all the other locations. Seeds from the establishment site will not be included in the mixture. Over time this will result in a population with wide adaptation. WP6: Genetic diversity analyses and trait/marker associations Responsible: UMB Participating: UMB and Univ of Aarhus Timeframe: (Daugstad, K 2011 On-Farm Conservation of the Forage Species Timothy, Meadow Fescue and Red Clover: Generation of New Landraces in Norway. In: Agrobiodiversity Conservation: Securing the Diversity of Crop Wild Relatives and Landraces (Editors: Nigel Maxted, Ehsan Dulloo, Brian V. Ford-Lloyd, Lothar Frese, José M. Iriondo, Miguel A.A. Pinheiro de Carvalho). In press 7

8 The aims of this work package is to; (a) genotype the accessions using a suitable marker system (preferably SNPs Single Nucleotide Polymorphisms), (b) estimate the genetic distance between the accessions, and (c) establish marker-trait associations (second phase). The molecular marker data will be generated from 200 accessions, selected on the basis of wide geographic distribution, using the individual plants per accession distributed for phenotyping at the four locations (WP4). Leaf samples will be taken at the time of distribution of plants from DLF, dried in silica gel and sent to Norway, and DNA extraction will be based either on single plants or on several bulks of equal amounts of dried leaf materials from the individuals. The SNP genotyping will be done using a high-throughput genotyping-by-sequencing strategy (GBS) 4 of pooled DNA modified at Aarhus University (Dr. Torben Asp). Equal amounts of DNA from the individual plants per accession will be pooled and used for construction of barcoded GBS libraries based on genome complexity reduction with a restriction enzyme. The 200 pooled and barcoded GBS libraries will be sequenced on an Illumina HiSeq2000 at Aarhus University. The approach is simple, quick, extremely specific, highly reproducible, and may reach important regions of the genome that are inaccessible to sequence capture approaches. The number of SNPs per pooled sample is determined by the restriction enzyme used for complexity reduction and will in such materials range from 10,000 up to 1,000,000. The genetic distance between the accessions and marker-trait associations will be established based on allele frequencies between the 200 pooled samples. Statistical tools for developing marker-trait associations based on allele frequencies in outbreeding species are currently being developed at Aarhus University in the Danish project Genomic selection in grasses, and will be available to be used in the project. The project leader will apply for a PhD student from UMB to work on this project and if this succeeds this will be an in kind contribution from UMB of approximately 750,000 DKK per year. Work for the next phase of the project (2014-?) In this phase of the project the phenotypic data generated in WP4 will be merged with the genotypic data in order to establish marker-trait associations (WP10). The haplotype-trait associations established from phenotypic data of single plants will be validated by genotyping and tracing of haplotypes in the broad breeding population established in WP5. Association of haplotypes with the trait data scored in the field tests undertaken at different locations in WP7 will also give valuable information for future improvement of specific traits and development of training populations for genomic selection. Genotyping for detecting allelic/haplotype shifts will be performed on the initial broad breeding population (WP5) and in populations generated after selection in the fields at the different locations (WP8). This will give valuable information about markers associated with adaptation, especially winter survival. 4 Elshire RJ, Glaubitz JC, Sun Q, Poland JA, Kawamoto K, et al A Robust, Simple Genotyping-by-Sequencing (GBS) Approach for High Diversity Species. PLoS ONE 6(5): e doi: /journal.pone

9 Work packages planned to start after WP7: Testing of plant materials from WP1 and WP2. The next step would be the actual test under different growth conditions in the Nordic countries. It is foreseen that field tests will be established at one location in Denmark, Finland, Estonia and Iceland, and at 1-2 locations in Norway and Sweden. The seed will be sown in small replicated plots and yield measurements, observations on winter survival, spring growth, development, and diseases conducted. The plots have to be observed in and ideally until spring 2017 to obtain good observations on winter survival after three winters. Sampling will also be done for detailed physiological and molecular analyses of cold acclimation, forage quality, snow mould resistance (Microdocium nivale). WP8: Establish the broad breeding population in different growing conditions The broad breeding population (WP5) will be established at locations representing different climatic conditions. The plots will be subjected to harvesting regimes representative for the locations, cut for forage production for 2 years and allowed to cross-pollinate the third year for producing a new population. Plots will be re-established from seeds of the new population and a new cycle of selection initiated. It is also possible to recombine this over locations in order to create a breeding population with wider adaptation. WP9: Test the accessions from WP1 and WP2 for winter survival (frost) and diseases resistance in lab tests. WP10: Identify marker-trait associations See description under WP6 9

10 Fig. 2. Flow-chart of the PPP for pre-breeding programme in perennial ryegrass (Lolium perenne L.) WP1 Requesting seed WP2 WP3 WP4 Seed multiplication Cultivar trials Phenotyping Broad breeding population Genotyping, diversity WP5 WP N, DK DK N, S, IS FIN, EE N, DK, S, FIN N, DK 2013 WP N, S, DK, IS FIN, IS, EE WP WP8 WP N, S, DK, IS FIN, IS, EE N, S, DK, IS FIN, IS, EE WP10 10

11 Table 2. Cost for the different WPs in 2012 (1000 DKK) Inst. / company Adm 1 Travel WP1 WP2 WP3 WP4 WP5 WP6 Sum Nordgen 46.0 UMB Graminor Lantmännen DLF Univ. of Aarhus Boreal Agr. Univ. of Iceland Jõgeva SUM ² ,175.2 ¹ UMB: project administration (100), hours used for two meetings á year for two days (18); other partners: hours used for two meetings á year for two days ²Estimated total costs for two project meetings and 10 persons; will be handled centrally (project adm). Travel costs for Jõgeva Plant Breeding Institute are included. Table 3. Cost for the different WPs in 2013 (1000 DKK) Inst. / company Adm 1 Travel WP1 WP2 WP3 WP4 WP5 WP6 Sum UMB Graminor Lantmännen DLF Univ. of Aarhus Boreal Agr. Univ. of Iceland Jõgeva SUM ² ,968.5 ¹ UMB: project administration (75), hours used for two meetings á year for two days (18); other partners: hours used for two meetings á year for two days ²Estimated total costs for two project meetings and 10 persons; will be handled centrally (project adm). Travel costs for Jõgeva Plant Breeding Institute are included. 11

12 Table 4. Total costs 2012, company contribution and public funding (1000 DKK) Institute / company Total costs Company contribution³ Public funding Nordgen UMB Graminor ⁴ Lantmännen DLF Univ. of Aarhus Boreal Agric. Univ. of Iceland Jõgeva Plant Breeding Institute 9.0¹ 0 Travel costs 150.0² TOTAL 1, ¹ Not included in the total costs; ² Including travel costs for Jõgeva Plant Breeding Institute; ³ DLF 30%, Graminor 30%, Lantmännen 20%, Boreal 20%; ⁴ Negative sum indicate cash contribution to the project from the company. Table 5. Total costs 2013, company contribution and public funding (1000 DKK) Institute / company Total costs Company contribution³ Public funding UMB Graminor ⁴ Lantmännen DLF Univ. of Aarhus Boreal Agrcultural Univ. of Iceland Jõgeva Plant Breeding Institute 22.0¹ 0 Travel costs 150.0² TOTAL 1, ¹ Not included in the total costs; ² Including travel costs for Jõgeva Plant Breeding Institute; ³ DLF 30%, Graminor 30%, Lantmännen 20%, Boreal 20%; ⁴ Negative sum Minus sign indicate cash contribution to the project from the company. 12

13 Table 6. Total costs 2012 and 2013, company contribution and public funding (1000 DKK) Institute / company Total costs Company contribution³ Public funding Nordgen UMB Graminor ⁴ Lantmännen DLF Univ. of Aarhus Boreal Agrcultural Univ. of Iceland Jõgeva Plant Breeding Institute ¹ 0 Travel costs ² TOTAL ¹ Not included in the total costs; ² Including travel costs for Jõgeva Plant Breeding Institute; ³ DLF 30%, Graminor 30%, Lantmännen 20%, Boreal 20%; ⁴ Negative sum Minus sign indicate cash contribution to the project from the company. 13