Ulva Biobanking, Phenotyping and Genotyping: The path ......Ulva Biobanking, Phenotyping and...

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?


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:


Natural variation: Strain collection

400 Individual laminar Ulva specimens from Ireland, the Netherlands, Scotland, France, Spain and Portugal

From intertidal

& subtidal areas

(snorkelling).


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


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


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


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



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




Fort et al, Plant physiology 2019 and unpub.

~4-fold difference in growth




~5-fold difference in protein content




~10-fold difference in starch

>demonstrate the potential to increase yield(s) following strain selection.

Also large differences in nitrate accumulation, pigments…




Each dot corresponds to 1 strain analysed


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


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…


Growth potential of Ulva strains at pilot scale


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:


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)


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


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


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


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


Genotyping: distribution in sampled areas

295 strains


Genotyping: distribution – addition of NCBI entries after curation

Fort et al, under review


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


Genotyping: distribution – U. lactuca

U. lactuca is absentfrom the North East Atlantic coast


Genotyping: distribution – species not really spread worldwide


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



CAPS assay:Each Ulva foliose species displays a specific pattern



CAPS assay:Can be applied to bulk biomassApplications for food and aquaculture


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


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!!


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

Ulva Biobanking, Phenotyping and Genotyping: The path ......Ulva Biobanking, Phenotyping and...

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