Progress in genomics applications in investigating abiotic stresses influencing perennial forage and biomass grasses

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1 Progress in genomics applications in investigating abiotic stresses influencing perennial forage and biomass grasses Dr. Susanne Barth Teagasc Crops Research Centre Oak Park Carlow Irland

2 Global challenges Improving productivity (dry matter yields) Adapting grasslands to changing environmental conditions - drought - cold - salinity - flooding - nutrient uptake and usage (N, P, K) Loss of diversity Soil erosion Land loss to other land uses Climate change

3 GrassMargins EU FP7 KBBE 2011 Aims to develop perennial rhizomatous grasses to enhance biomass production from marginal lands (Dactylis & Phalaris are candidate species) Marginal land Poor climate Low fertility Degraded or polluted High salinity Drought Poor drainage (wetland) grassmargins.com

4 Drought stress Globally drought induced losses in crop yield exceed losses from all other causes. Solutions: breeding and management practices (hormones or osmoprotectants) Tolerance is often associated with the accumulation of solutes. F83, 10% damage F121, 75% damage

5 Water logging Flooding lesser studied stress, although becoming more pronounced under climate change. Oxygen concentrations in the root zone reduced. Phalaris arundinacea has been reported to be adopted to flooding conditions (Etherington 1984), and also genotypic differences in cocksfoot were found. McLeod et al demonstrated that Festulolium cultivars can reduce significantly run-off during flooding compared to Lolium and Festuca cultivars.

6 Phalaris arundinacea (Reed Canary Grass) Water margins and wetlands 2x, 4x and 6x ploidy Dactylis glomerata (Cocksfoot) Range of habitats 2x, 4x ploidy Both are C3, perennial, rhizomatous, wind pollinated, outbreeders. Temperate Eurasian origin.

7 Flooding stress Flooding in submerged tubes Harvest of Phalaris after 4 weeks complete flooding

8 Genotype-dependent response to flooding

9 Waterstress experiment Treatment effects monitored by Soil moisture content Plant height Chlorophyll content Leaf water content Electrolyte leakage biomass

10 Soil water content between treatments d1 : begin of differential watering

11 Plant growth between treatments d1 : begin of differential watering

12 References: assemblies from Illumina reads All reads of controls and treatments were combined: assembly from 800 M (Phalaris) and 1bn (Dactylis) reads from a single cultivar Transcripts < 200 bases were discarded Highly similar reads (>95%) were combined into one transcript sequence Only the longest sequence of each gene was kept in the final result Resulting Transcriptomes with ~60000 (Phalaris) and ~69000 (Dactylis) transcripts of >200 b length

13 Phalaris annotation: top hits by species

14 Dactylis annotation: hits by species

15 RoDEO: Differential expression Detection Robust Differential Expression Operator Nonparametric Framework for detecting differential expressions in RNA-Seq data Nonparametric framework (agnostic to platforms, read biases etc) Competitive with existing methods & handles additional scenarios (evaluated on simulation, plant and human data) Outperforms other methods in the elite-top genes Resilient, i.e., works well also in the absence (technical/biological) replicates bayseq Assumes distribution for counts Requires replicates -ve binomial edger -ve binomial X sseq -ve binomial RoDEO None (non-parametric)

16 Water logging and drought transcript study in Phalaris arundinacea 2 (Intersection of DOWN and UP sets is empty) (Intersection of DOWN and UP sets is empty) 4 Comparing time 0 with time 2 (before stress and last time point at stress) Robust DOWN-regulated* transcripts (intersection of red & green) are: 1) DOWN-regulated for stress 0 vs. stress 2 (red), AND 2) DOWN-regulated for control 2 vs. stress 2 (green) Haiminen et al. 2014, BMC Genomics [*similarly for UP-regulated]

17 Salt stress: C3 grasses Salt stress in Europe is mainly a problem in Southern and SouthEastern regions, but will increase with climate change. Didion et al., unpublished data Tall fescue > Festulolium > Cocksfoot > Reed canary

18 Salt stress: Miscanthus 28 Miscanthus accessions plants potted in soil (15-20 pots with plants of the same clone) Strictus, H7 Didion et al., unpublished data

19 Cold tolerance Vast areas of marginal land in the cool-temperate climatic zone (Eurasia and North-America) could be potentially cultivated to meet a future demand for bioenergy and forage crops. Gudliefsson et al investigated 10 species: Phleum pratense & Poa pratensis > Dactylis glomerata & Phalaris arundinacea M. sinensis no. KK39 highest rate in daily leaf growth during the cool period, M. sacchariflorus, EMI3 highest growth rate during warm period. Most M. sacchariflorus genotypes showed a high daily growth rate during the cool period. Jiao et al., 2016 Annals of Botany

20 A genespace assembly of 10th generation inbred PRG line using Illumina shotgun sequencing Range of Illumina library types Paired-end (PE): up to 500bp insert size Mate pair (MP): 3Kbp insert size Long-jumping distance (LJD): 8Kbp, 20Kbp, 40Kbp Mostly 100nt read lengths PE libraries form core of the assembly Sequencing coverage around 115x Core assembly using CLC Genomics Workbench MP and LJD used for scaffolding Sequencing coverage around 100x Scaffolded using SSPACE

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22 Pst1 2 channels HiSeq2000 SNP Framework PAVs fitted Presence/Absence Variants Error correction by imputation

23 An ultra-high density map of perennial rygrass Total no. markers 10,353 Total SNPs 3,105 Total PAVs 7,248 Markers/LG Total unique bins 1865 Unique bins/lg Map length 952.6cM Scaffolds Anchored 4767 Total Length anchored ~200Mb Anchored/marker ~20kb Velmurugan et al. (2016) An ultra-high density genetic linkage map of perennial ryegrass (Lolium perenne) using genotyping by sequencing (GBS) based on a reference shotgun genome assembly. Annals of Botany, in press

24 GS and GWAS in perennial ryegrass breeding GS for spaced plant selection in breeding programme Training population of 1800 plants (GWAS population) 30 diploid ryegrass populations (10 cultivars, 8 full-sib, 8 half-sib, 4 ecotypes). 60 genotypes from each population clonally propagated Replicated trials for three management regimes Targets: quality, flowering time, reheading, growth habit, rust resistance, persistency GBS with ApeK1

25 Conclusions Conventional breeding in forages and bioenergy grasses has room for crop improvement compared to other more intense bred crops like maize For outbreeding forage crops improvement by marker assisted selection, including genome wide association mapping or genomic selection is advancing Advances in transcriptomes of lesser advanced grasses are being made and are an entry level for other omics work in less characterized grass genomes Different grass species suit different locations and stress conditions better: Good choice of grasses and also mixture of species necessary

26 Acknowledgements Phalaris, Dactylis, Festuca and Miscanthus work financed by EU FP7 GrassMargins project (FP7 KBBE ) Lolium work financed by Teagasc and the Irish Department of Agriculture, Fisheries, Food and the Marine Stimulus Fund (11/S/109) Partners in the GrassMargins project Bioinformatics group James Hutton Institute Teagasc colleagues IBM Research collaborators