Investigations into biomass yield in perennial ryegrass (Lolium perenne L.) Ulrike Anhalt 1,2, Pat Heslop-Harrison 2, Céline Tomaszewski 1,2, Hans-Peter Piepho 3, Oliver Fiehn 4 and Susanne Barth 1 1 2 3 4
Contents of presentation Ryegrass, economy, breeding objectives Background of inbred plant material and population F2 genetic map Phenotypic experiments Results phenotypic and QTL studies Metabolite approach Further directions
Perennial ryegrass Irish economy Irelands agricultural product 2001 agricultural land 2003 Perennial ryegrass is one of the most productive forage species in Irish grasslands Source: Compendium of Irish statistics 2004
Breeding objectives in ryegrass Improved yield Improved forage quality Persistency Resistance to crown rust and other diseases Improved abiotic stress tolerance Growth in shoulder periods of the season
Breeding for biomass Forage for grazing, silage and hay with high feeding value Renewable energy (biogas, ethanol and combustion) for bioelectricity Fibre production for insulation material in biorefineries
Background of plant material Wildtype Lolium perenne is allogamous, highly heterozygous and self-incompatible Parental lines derived from the experimental breeding programme of Dr. Vincent Connolly/Oak Park Inbred lines with 7-12 generations of inbreeding Inbred lines from different genetic backgrounds Festuca pratensis Lolium perenne 1968 Lolium perenne BC Lolium perenne BC S1 S7-S10 Parental inbred line 1982
pping population Cross of 2 inbred lines 2004 2005 No larger Festuca pratensis blocks in Hybridization probe: parental DNA (green) + 18S-26S rdna genes (red); Chromosomes DAPI (blue) F2 mapping population genotyped with 360 individuals 2008
Features of mapping population heterosis for biomass morphological and developmental traits: leaf shape, habit Different levels of crown rust resistance/ susceptibility ternal maternal Paternal paternal metabolites? Nutrient use efficiency Inbreeding depression heterosis
Construction of a F2 genetic map LG1 0.0 NF155**** LG2 0.0 15.1 19.0 51.3 LpSSR027 61.3 67.8 70.1 73.7 B1B6** rv0252 M16B**** LpACT15H3* 90.1 95.8 rv0327**** EacaMcac-415** LG3 0.0 EagcMcta-230**** 6.9 rv1133**** NF136*** 45.2 48.3 54.6 rv1117 rv1269 63.7 M15185 G04_059** 11.0 17.0 20.6 22.3 25.4 28.0 46.5 NF017** DLF25 G04_072* 18.0 rv0380* rv0262 30.5 34.8 39.3 41.1 G04_099*** 44.9 55.2 rv1412** 63.2 64.4 71.1 PRG PR14 rv0068 43.7 rv1131*** 50.5 54.9 57.8 62.3 66.2 68.7 rv0029*** B3B8** LPSSRH02F01*** rv1046 LpHCA18A2b** rv0360 63.5 84.6 88.6 rv0188 94.7 95.3 PR8**** 110.9 LG6 EagcMcta-140*** 0.0 22.8 26.6 30.5 34.7 NF142* LG5 0.0 0.0 9.6 12.0 14.1 LPSSRK142**** 21.0 25.3 B1A2**** rv0863**** rv0674*** G04_054**** EacaMcac-445 rv0062 34.9 LG4 rv0342**** rv1188 LpHCA18B12 rv1139 rv0562 rv1024 40.8 45.2 G04_043**** rv0739**** LpACTR1C5**** rv0307**** rv0983**** RV0171**** NF036**** rv0196**** LG7 0.0 EagcMcta-51**** 28.5 rv0134**** 34.5 EacaMcac-213**** Rye14**** 41.0 LPSSRH11G05**** 47.9 52.7 G04_002**** rv1411 B3A3 63.9 B1C8**** 75.9 LpSSR20**** 88.7 rv0717 EagcMcta-108**** EacaMcac-433 LpACT44A7 LPSSRK12E06 360 F2 genotypes 74 markers (SSR and AFLP) rker density: ø 8 cm p length: 595 cm Asterisks indicate segregation distortion (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001) Anhalt et al. (2008) TAG, 117: 297-306
Phenotypic experiments Alpha lattice design Incomplete block design (each block consist of eight F2 genotypes plus one check) 360 F2 genotypes and 45 checks (paternal, maternal and plants) Block with eight entries plus one check greenhouse and field experiments traits: (leaf width), (heading date), fresh weight, dry weight and dry matter
Phenotypic data - Greenhouse single plants three replications three harvests 2005-2006
Phenotypic data - Field 2006 to 2007 mini swards two replications four harvests
Results heritability heritability greenhouse field Fresh weight 0.8033 0.8833 Dry weight 0.9540 0.8838 Dry matter 0.7780 0.8614 Data were reproducible across all environments and replications
QTL - greenhouse and field (MQM) Greenhouse: MPH fresh weight: 99-182% BPH fresh weight: 115-223% MPH dry weight: BPH dry weight: 80-173% 113-219% Field: MPH fresh weight: (60 120%) BPH fresh weight: 52-772% MPH dry weight: BPH dry weight: (48-110%) 47-681% Parental inbred lines weak under field conditions!
% variation and gene action % variation explained: Fresh weight Dry weight greenhouse 26 32.6 field 24 30.7 Gene action: LG2 LG3 LG7 greenhouse additive additive (dominance) dominance field additive (dominance) dominance (additive) dominance
Continued mapping Example LG2 10 5 5 0 0 LPSSRK12E06 rv0188 rv0062 Loc-OS04g55260 LpHCA17C11 M15185 rv1212 Loc-OS04g55060 rv0959 G04_059 rv1282 rv1269 rv1117 G01-039 G04-053 NFF Loc- a136 Os04g54940 EacaMcac-415 G04_030 EacaMcac-445 rv0188 G04_030 M15185 G04_059 rv1269 rv1117 NF136 rv0062 EacaMcac- 445 EacaMcac-415 Further SSRs and rice STS markers added: Change of marker positions(*), marker density ( ) Previous QTL location * * p (cm) * p (cm) LPSSRK12E06 10
Progress in the RIL programme 2004 2005 All plant lines were generated from single seed descent Population consists of 400 genotypes 2008
Metabolites of parental and lines ternal, paternal and lines were grown in 2006 in 18 replicates each, and harvested 3x over the growth period (June, August, October) Total metabolites were extracted Analysed by GC-MS
Metabolites - results 253 metabolites were found, of those only 84 were known compounds Among the unknown metabolites the largest x-fold differences among /maternal and /paternal lines were found ximum up- and downregulation of metabolites: genotype maternal/paternal /maternal /paternal up regulation down regulation 11.3 19.2 5.7 10.5 22.2 6.5
Metabolite PCA across three harvests 10 maternal line 5 line 0 scr[2] PCA2 paternal line Clear separation according to lines -5 PCA1: 30% explained PCA2: 15% explained -10-15 -30-20 -10 PCA1 scr[1] 0 10
Further directions Fine mapping in the F2 population with ryegrass and rice markers Physical mapping of distances among QTL from ryegrass to rice Fine mapping in RILs Detailed metabolite analysis in F2 (under nutrient stress conditions: Dr. Stephen Byrne and Alexandre Foito, work underway) (mirna profiling)
Acknowledgements Dr. Vincent Connolly for inbred lines Ms Aurélie Guillard for metabolite extractions Numerous summer students for plant propagations and seed cleaning in the RIL programme Grass breeding and farm team at Oak Park