Ulva Biobanking, Phenotyping and Genotyping:
The path towards breeding
Ronan Sulpice, Antoine FortPlant Systems Biology lab
sulpice-lab.com
This project has received funding from the European Union’s Horizon 2020 research and innovation programme under Grant Agreement no. 727892 (GENIALG)[email protected]
This project has received funding from the European Union’s Horizon 2020 research and innovation programme under Grant Agreement no. 727892 (GENIALG).
Expansion of production is about cultivation, in Asia
Can we develop a large and sustainable seaweed aquaculture in Europe?
This project has received funding from the European Union’s Horizon 2020 research and innovation programme under Grant Agreement no. 727892 (GENIALG).
Towards Ulva spp. breeding
• Know what we work with. Species identification, extent of intra-species genetic variation
• Describe genetic / natural variation for growth and biochemical composition. Is strain selection worthwhile?
• Document environmental factors impacting growth and biochemical composition
• Keep for long term the strains.
> When achieved, we will have the tools for Marker Assisted / Genomic Selection
We need to:
This project has received funding from the European Union’s Horizon 2020 research and innovation programme under Grant Agreement no. 727892 (GENIALG).
Natural variation: Strain collection
400 Individual laminar Ulva specimens from Ireland, the Netherlands, Scotland, France, Spain and Portugal
From intertidal
& subtidal areas
(snorkelling).
This project has received funding from the European Union’s Horizon 2020 research and innovation programme under Grant Agreement no. 727892 (GENIALG).
Cryopreservation of thalli Ulva strains
WP2
Safeguard phenotyped/genotyped strains (and their symbionts) for future studies and breeding efforts
Survival of all strains tested when put back in culturCa 50% restarted vegetative growth, 50% sporulated
Protocol also tested at SAMS, SBR, CIIMAR and WUR
This project has received funding from the European Union’s Horizon 2020 research and innovation programme under Grant Agreement no. 727892 (GENIALG).
Cryo-preservation
Protocol slightly adapted from NUIG-protocol
One week after thawing thalli start sporulating
RGR for population on the left hand side: 20-20% per day
Protocol should further optimized for commercial purposes
This project has received funding from the European Union’s Horizon 2020 research and innovation programme under Grant Agreement no. 727892 (GENIALG).
Raspberry pi Pi CameraNetwork
Currently:- 9 remote-controlled Raspberry
pi computers- Image capture every 2 minutes
during daylight - Discs of Ulva thallus- Artificial seawater + nutrients- Can change environmental
conditions
Fort et al, Plant physiology 2019
Natural variation: Phenotyping
This project has received funding from the European Union’s Horizon 2020 research and innovation programme under Grant Agreement no. 727892 (GENIALG).
Raw image (every 2’) Segmentation Merging every 10’ 10’ merged image
ImageJ time-lapse Stacks (7 days-worth) Data analysis
Fort et al, Plant physiology 2019
Natural variation: Phenotyping
This project has received funding from the European Union’s Horizon 2020 research and innovation programme under Grant Agreement no. 727892 (GENIALG).
Throughput: 414 disks / run -> 23 strains with 18 disks per genotype> We can do strain selection fast and efficiently
Run time: 7 days
Data obtained / run:
• Growth rate (area and biomass-based)• Morphology (e.g. thickness)• Metabolites: soluble sugars, proteins, starch, pigments, amino acids, nitrates, nitrite, …. • C / N / S and ash content
Natural variation: Phenotyping
This project has received funding from the European Union’s Horizon 2020 research and innovation programme under Grant Agreement no. 727892 (GENIALG).
Natural variation: Phenotyping
Fort et al, Plant physiology 2019 and unpub.
~4-fold difference in growth
This project has received funding from the European Union’s Horizon 2020 research and innovation programme under Grant Agreement no. 727892 (GENIALG).
Natural variation: Phenotyping
Fort et al, Plant physiology 2019 and unpub.
~5-fold difference in protein content
This project has received funding from the European Union’s Horizon 2020 research and innovation programme under Grant Agreement no. 727892 (GENIALG).
Natural variation: Phenotyping
Fort et al, Plant physiology 2019 and unpub.
~10-fold difference in starch
>demonstrate the potential to increase yield(s) following strain selection.
Also large differences in nitrate accumulation, pigments…
This project has received funding from the European Union’s Horizon 2020 research and innovation programme under Grant Agreement no. 727892 (GENIALG).
Natural variation: Phenotyping
Fort et al, Plant physiology 2019 and unpub.
Each dot corresponds to 1 strain analysed
This project has received funding from the European Union’s Horizon 2020 research and innovation programme under Grant Agreement no. 727892 (GENIALG).
Where to find the best Ulva strains? Green tides might be the place
Fast growth, high protein and low carbohydrate content in green tide strains could make them important for aquaculture.
Also could partly explain the aggravation of green tide events since the 70’s…
Fort et al, STOTEN 2019
Light quality - phenotyping Ulva local strains
Isabel Azevedo, Helena Amaro, Rosa Melo, Isabel Sousa Pinto
This project has received funding from the European Union’s Horizon 2020 research and innovation programme under Grant Agreement no. 727892 (GENIALG).
Light quality trials- phenotyping Ulva local strainsMethodology
50 µmolphonton. m-2.s-1
Green LED Red LED Blue LED White LED
Ulva laetevirens(from Belinho)
#1- Effects of light quality on growth, pigments production and antioxidant capacity
GFR WFR
With 5% of Far-Red
#1- Effects of light quality on growth, pigments production and antioxidant capacity
Major conclusions
✓ Using W, R and GFR is possible to modulate Ulva laetevirens production and bioactive abilities, promising to nutraceutical market.
✓ Light quality affects Ulva laetevirens growth and composition
50 µmolphonton. m-2.s-1
Green LED Red LED Blue LED White LEDUva rigida(from Belinho)
#2
Ulva rigida(from Amorosa)
Ulva laetevirens(from Matosinhos)
Does light quality have different effects on different Ulva species and strains?
Biochemical composition
Pigments profile and contentTotal carbon hydrates
Total phenolic compounds Proteins content
Fluorescent light
Light quality trials- phenotyping Ulva local strainsMethodology
Major conclusions
Higher under R Highest in terms of species and light qualities
In general terms Ulva xanthophylls are induced by G, F and carotenes by R
Highest content in carotenoids of Belinho strain under G, 20% more than Amorosa strain, namely 2-fold the content in lutein
✓ Light quality affects the different Ulva strains pigments production and growth Seems to be strain dependent
Uva rigida from Belinho Ulva rigida from Amorosa Ulva laetevirens from Matosinhos
Higher under GGrowth
Carotenoids
Higher equally under G, R and W
Antioxidantactivity
undergoing…
Biochemicalcomposition undergoing…
This project has received funding from the European Union’s Horizon 2020 research and innovation programme under Grant Agreement no. 727892 (GENIALG).
Growth potential of Ulva strains at pilot scale
This project has received funding from the European Union’s Horizon 2020 research and innovation programme under Grant Agreement no. 727892 (GENIALG).
Growth potential of Ulva strains
• Growth analyses of Ulva populations collected at 13 sites 2017-2019• N-addition experiments 2019-2020• Chemical composition: amino acids, protein, nitrogen, starch, ash• Bio-banking & cryo-preservation of collected strains
Activities @ WUR:
This project has received funding from the European Union’s Horizon 2020 research and innovation programme under Grant Agreement no. 727892 (GENIALG).
Genotyping: Intra- and inter-species variations.
1) Ulva’s phylogeny> Species/sub-species/hybridization events
2) Detect genetic variation among Ulva strains associated with growth/metabolic variations
> Identify genes/locus: Genome wide association studies> Genetic markers that predict performance: Genomic selection
> Whole Genome Sequencing of 110 of the phenotyped Ulva strains (Illumina HI-Seq, 15X coverage)> Generated 1 assembly genome using MinION (nanopore) and another ongoing (PacBio)
This project has received funding from the European Union’s Horizon 2020 research and innovation programme under Grant Agreement no. 727892 (GENIALG).
Genotyping: DNA extraction for Next Generation Sequencing
Unlike barcoding, next-generation sequencing require high amounts of good quality DNA
> modified magnetic
beads extraction protocol
for best DNA quality
(also 3x cheaper than
column kits)
> Efficient for kelp,
seagrass, red seaweeds,… Fort et al., Algal Research 2018
This project has received funding from the European Union’s Horizon 2020 research and innovation programme under Grant Agreement no. 727892 (GENIALG).
Genotyping: Inter-species variations are large for organelles
ChloroplastChloroplast Mitochondria
Species
Number of
SNPs / kb
Versus
U.laetevirens
(CDS)
Number of
SNPs / kb
Versus
U.laetevirens
(CDS)
U.australis 82 196
U.fenestrata 79.8 194
U.rotundata 91.5 222
U.gigantea 35.3 86.1
U.rigida 6.1 5.6
An issue for Genome Wide Association Studies and genomic selection: need to focus on 1 species
This project has received funding from the European Union’s Horizon 2020 research and innovation programme under Grant Agreement no. 727892 (GENIALG).
Genotyping: Intra-species variations are low
Ulva’s phylogeny> Species/sub-species/hybridization events
110 strains, representing 6 Ulva species sequenced by IlluminaAssembly of chloroplast and mitochondrial genomes of the 6 species
Chloroplast Mitochondria
Species Strains SNPs/Kb SNPs/Kb
U.australis 16 1.98 ± 0.66 1.05 ± 0.9
U.fenestrata 11 0.12 ± 0.07 0.05 ± 0.05
U.rotundata 6 0.09 ± 0.09 0.04 ± 0.4
U.gigantea 10 0.38 ± 0.15 0.26 ± 0.09
U.rigida 29 0.18 ± 0.14 0.03 ± 0.05
U.laetevirens 38 1.14 ± 0.7 1.36 ± 0.7
U. Fenestratachloroplast
Fort et al, J.phycol. 2020
This project has received funding from the European Union’s Horizon 2020 research and innovation programme under Grant Agreement no. 727892 (GENIALG).
Genotyping: presence of hybrids in the wild?
F1: Ulva rigida and laetevirens can hybridise
Also we found F2s in another study But only about U. rigida and U. laetevirens, and it is very rare
This project has received funding from the European Union’s Horizon 2020 research and innovation programme under Grant Agreement no. 727892 (GENIALG).
Genotyping: distribution in sampled areas
295 strains
This project has received funding from the European Union’s Horizon 2020 research and innovation programme under Grant Agreement no. 727892 (GENIALG).
Genotyping: distribution – addition of NCBI entries after curation
Fort et al, under review
This project has received funding from the European Union’s Horizon 2020 research and innovation programme under Grant Agreement no. 727892 (GENIALG).
Genotyping: distribution – addition of NCBI entries after curation
Main messages for large foliose Ulva:
1. 21% of entries are misannotated, incl. 65% of U. lactuca
2. U. armoricana and U. scandinavicaare synonymous to U. laetevirens
This project has received funding from the European Union’s Horizon 2020 research and innovation programme under Grant Agreement no. 727892 (GENIALG).
Genotyping: distribution – U. lactuca
U. lactuca is absentfrom the North East Atlantic coast
This project has received funding from the European Union’s Horizon 2020 research and innovation programme under Grant Agreement no. 727892 (GENIALG).
Genotyping: distribution – species not really spread worldwide
This project has received funding from the European Union’s Horizon 2020 research and innovation programme under Grant Agreement no. 727892 (GENIALG).
Genotyping: high throughput and bulk biomass analysis
CAPS assay:Use of restriction enzymes after PCR amplification of the ITS1
Cheap and fast
> Species identification without sequencing
Fort et al, under review
This project has received funding from the European Union’s Horizon 2020 research and innovation programme under Grant Agreement no. 727892 (GENIALG).
Genotyping: high throughput and bulk biomass analysis
CAPS assay:Each Ulva foliose species displays a specific pattern
This project has received funding from the European Union’s Horizon 2020 research and innovation programme under Grant Agreement no. 727892 (GENIALG).
Genotyping: high throughput and bulk biomass analysis
CAPS assay:Can be applied to bulk biomassApplications for food and aquaculture
This project has received funding from the European Union’s Horizon 2020 research and innovation programme under Grant Agreement no. 727892 (GENIALG).
Conclusion
➢ Large phenotypic variation (ca 5X) justify strain selection prior cultivation
➢ Seven foliose Ulva species identified. Lot of misannotations and requirement for a change in legislation (no U. lactuca in the areas we sampled).
➢ Cheap and fast assay for Ulva spp. ID, incl. in bulk will facilitate traceability and certification
➢ Cryopreservation of thalli is possible and will facilitate the creation of biobanks
This project has received funding from the European Union’s Horizon 2020 research and innovation programme under Grant Agreement no. 727892 (GENIALG).
Future avenues➢ Genome wide association mapping and genomic selection is possible BUT:Species are too different so need to focus
➢ Investigate growth and metabolic response to different abiotic conditions for efficient on-shore farming using elite strains
➢ Validation of phenotypic data in aquaculture settings
➢ In Galway: creation of a graph database containing genetic and phenotypic data of strains cryopreserved to support Ulva strain selection. Will open to public
>>still a lot to do!!
This project has received funding from the European Union’s Horizon 2020 research and innovation programme under Grant Agreement no. 727892 (GENIALG).
Acknowledgements
Plant systems biology lab
• Dr. Antoine Fort
• Dr. Jose Maria Fariñas-Franco
Now GMIT
• Dr. Masami Inaba
• Dr. Marcus McHale
• Ms. Clara Simon
• Ms. Charlene Linderhof
• Ms. Ines Coca Tagarro
Prof. Michael Guiry
Nanopore genome
• Prof. Bjoern Usadel
• Prof. Philippe Potin
• Dr. Kevin Cascella
An many others involved in other aspects not covered today
Pristine Coasts ltd, providing help for species ID
GenialG partners