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Control of the reproductive cycle in Azolla filiculoides

2015-2016 Internship

Thursday 08/09/2016Georgos Zangos

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Azolla filiculoides Lam

Azolla filiculoides is a fern that harbors symbiotic nitrogen-fixing cyanobacteria.

Azolla grows free-floating on freshwater. Image from the Ecolink website.http://ecolinc.vic.edu.au/sites/default/files/uploaded_files/discovering_wetlands/gallery/view/141-1073.html

Azolla (left) and its endosymbiont Anabaena (right). Image courtesy of the University of Wisconsin’s Department of Botany website.http://botit.botany.wisc.edu/Resources/Botany/Bacteria/Anabaena/

http://theazollafoundation.org/azolla/the-azolla-anabaena-symbiosis-2/

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It occurs in ponds, ditches and paddy fields of warm-temperate and tropical regions throughout the world (Lumpkin 1980)

http://www.au.all.biz/azolla-filiculoides-red-azolla-g13190#.VmbeOLgrLcs

Azolla filiculoides Lam

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Azolla filiculoides Lam

The agronomic potential of Azolla has long been recognized in Southeast Asia as a biological nitrogen fertilizer for rice cultivation (Moore, 1969).

http://anupamfolkrice.blogspot.nl/2014/09/mr-purnendu-basu-agriculture-minister.html

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Azolla filiculoides Lam

Azolla was also used as a protein supplement for animal feeding (Becerra et al., 1990), (Leterme et al., 2010)

http://dhartiputrakisansewa.blogspot.co.uk/

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Azolla filiculoides Lam

or as biomass for biofuel, partially replacing soybean in tropical and temperate countries with sufficient water (Hossain et al., 2011).

https://kenaseka.wordpress.com/

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Control over Azolla sexual reproduction will be of paramount importance for disseminating existing varieties or breed new varieties.

The Azolla genome has been sequenced and a 37649 unigene database covers metabolism, cellular processes and regulatory networks.

165 unigenes were similar to Arabidopsis thaliana genes involved in flowering.Brouwer et al. 2013

miRNA 156 and 172 that control the transition to sexual development in seed plants have been reported in ferns but their role in the induction of sporulation remains unknown.

The repertoire of fern miRNA is also entirely unknown.

Domestication of Azolla

9http://www.phytosystems.ulg.ac.be/florid/networks/aging/microrna_biosynthesis

miRNA

10 http://www.phytosystems.ulg.ac.be/florid/networks/aging/mir156_functions

miRNA 156 & miRNA 172

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http://www.phytosystems.ulg.ac.be/florid/networks/aging/mir156_functions

miRNA 156 & miRNA 172

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Nitrogen in the medium represses sporulation.

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Nitrogen in the medium represses sporulation.

sample pH at DAY36 -N I 6,00 -N II 6,09 -N III 6,21 +N I 3,72 +N II 3,66 +N III 3,62 NH4 I 4,04 NH4 II 3,73 NH4 III 3,86 NO3 I 7,14 NO3 II 7,38 NO3 III 7,57

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Arabidopsis response to FR and TL light

(n= 10-20) (n= 15-21)

(n= 42)

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Bioinformatic analysis

mirna folding and prediction

Small RNA extraction

Small RNA Raw reads

SmallRNA library preparation and sequencing

Removal of low quality reads, sequences without primer and barcode

Trimmed Sequences

Mapped Reads

Map to the genome

miRNA candidates

identify homologous miRNAs

Conserved miRNA

Novel miRNA

Exclude false candidates,Introns, exons, other non-coding-RNA

CAAGAGAAACGCAAAGAAACUGACAGAAGAGAGUGAGCACACAAAGGCAAUUUGCAUAUCAUUGCACUUGCUUCUCUUGCGUGCUCACUGCUCUUUCUGUCA

200 nt extention

Mir/miR* up to 4 mismatches, 2 nt overhang in each armmax 1 bulge of 2nt (Mayers et al., 2008)

MFE < -20 (Bonneet et al., 2004)

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Bioinformatic analysis test

Tests: 8 trimmed small-RNA libraries generated by four developmental stages of leaves and siliques (Thatcher SR, et al. 2014) and the Arabidopsis genome.

Short-stack (Axtel et al., 2013) – web based Automatically trims reads,

map reads to the genome and predict miRNA candidates

18 miRNAs candidates predicted and uploaded to miRBase for homology resulting in 16 mature miRNA / 427 reported in miRBase (100% identical)

sharp prediction that delivers high probability positive miRNAs but at the cost of very low miRNA discovery

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Bioinformatic analysis test

545 miRNAs candidates predicted bu miREAP and subjected to miRBase for homology resulting in

21 mature miRNA / 427 reported in miRBase (100% identical)

A more flexible set of toolsBowtie mappingmiREAP folding & prediction andBlastn homology

Higher number of predicted miRNAsSafe to proceed with

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A scheme of conditions was planned for differential miRNA sequencing.Triplicates of each condition was assessed

smallRNA libraries preparation

FR TL

Azolla+cyano/-N +cyano/-N+cyano/+N-cyano/+N

Arabodopsis

Azolla plants constantly kept under TL ligh were transferred to FR lightSamples were collected 7 days later 0800 hArabidopsis seeds were sawed to adjusted ½ MS medium (no sugars, no vitamins, no intibiotics) and were collected 7 days after germination 0800 h

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small-RNA sequence

5’ Adapter ligation

cDNA Synthesis

PCR amplification 15 cycles

PCR PrimersSample Barcode

3’ Adapter ligation

library purification 8% PAGE gel

smallRNA libraries preparation

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160 bp140 bp

smallRNA libraries preparation

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smallRNA libraries preparation

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PCR amplification

Raw read

Clustering Sequencing

Next Generation Sequencing 1 x 75 bp reads single end Illumina NextSeq500 was used

smallRNA libraries preparation

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Bioinformatic analysis

Small RNA extraction

Small RNA libraries 285,219,714 raw reads

SmallRNA library preparation and sequencing

Removal of low quality reads, sequences without primer and barcode using Trimomatic

Trimmed Sequences 276,904,904 reads

165,891,928 total reads21,935,456 unique reads

Discart sequences longer that 26 nt and shorter than 18nt

93,867,190 (83.69%) Azolla52,285,210 (97.30%) Arabidopsis

mapped reads

Map to the genomes using topHat

11,304 Azolla and2,202 Arabidopsis miRNA candidates

mirna prediction using miRa

Blast against miRBase to identify homologous miRNAs

76,111 Azolla and9,654 Arabidopsis miRNA

candidates

13 conserved miRNAs in Azolla and35 in Arabidopsis

Exclude false candidates

mira name chromosome start end Mira output real sequence

precursor_2570_0_minus 2 6566469 6566497 GTGAAATCGGAGAGGGAATTCGTCAGCG CAAAATCGCCAACGAATTTCCTCTCCGA

GAGAGGAAATTCGTTGGCGATTTT TGACGAATTCCCTCTCCGATTTCA

precursor_3395_3_plus 5 14154330 14154354 TTGACCCATGAATTTTGACCCATT TTGACCCATGAATTTTGACCCATT

TTAGAGATTTTAGTTGGTTTAA TTAGAGATTTTAGTTGGTTTAA

precursor_2621_0_plus 2 6612085 6612110 AGAGCCGGTTGTTGGAGAGTTGGTC AGAGCCGGTTGTTGGAGAGTTGGTC

mira name chromosome start end Mira output real sequence

precursor_2570_0_minus 2 6566469 6566497 GTGAAATCGGAGAGGGAATTCGTCAGCG CAAAATCGCCAACGAATTTCCTCTCCGA

GAGAGGAAATTCGTTGGCGATTTT TGACGAATTCCCTCTCCGATTTCA

precursor_135_8_minus 3 1924211 1924236 AAGTGTACGAATCAAACCGTGAAAA AGTGTACGAATCAAACAGTGAAAAA

miR172 not discovered

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Bioinformatic analysis

Small RNA extraction

Small RNA libraries 285,219,714 raw reads

SmallRNA library preparation and sequencing

Removal of low quality reads, sequences without primer and barcode using Trimomatic

Trimmed Sequences 276,904,904 reads

165,891,928 total reads21,935,456 unique reads

Discart sequences longer that 26 nt and shorter than 18nt

72,474,851 total mapped reads15,239,874 unique mapped reads

Map to the genomes using Bowtie

11,305 Azolla and20203 Arabidopsis miRNA

candidates

mirna prediction using miREAP

Blast against miRBase to identify homologous miRNAs

11,305 Azolla and2203 Arabidopsis miRNA

candidates

21 conserved miRNAs in Azolla and33 in Arabidopsis

Exclude false candidates

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Bioinformatic analysis

conserved miRNA number of members sequence miR156 1 GGCUGUGCUCUCUCUCUUCUG miR159 1 UUGGACUGAAGGGAGCACCAC miR160 1 AUGGCAUCCAGCGAACCGGCAUCCG miR172 1 GUGAGAAUCCUGAUGAUGCUGC miR319 1 AUUGGACUGAAGGGAGCUGCUU miR482 2 GUGGGUGGGAUGGGAAGGAUU miR529 1 GCUGUGCUUUCUCUCUUCUCAU miR845 1 AAGCUGUGAUACCAAUUGUUGGGA miR865 1 UCAAUUAUAUCCACAAAUCAUCC miR1435 1 UAUAAACAGGUUUGACUUCAAGGU miR2102 2 CGAUGGUGACCGGUACCGGUGGC miR2630 1 UGGUUUUGGUUUUGGUUUUUC miR5675 2 UUCCUUUGUUGUCGUGCUCUUUCCGU miR6300 2 CUCACCACAAUACUGCAACGACCU miR8175 1 GUUCGAUCCCUGGCAACGGCGC

15 conserved mature miRNAs encoded by 19 loci were discovered in Azolla including miR156 & miR172 33 mature miRNAs were discovered in Arabidopsis samples (not miR172)

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miR156 & miR172 are present in Azolla

a

b

miR172

miR156

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Quantification

Each samples’ sequence was aligned - mapped to predicted pre-miRNAs using Bowtie.

Quantities were normalized by dividing to the Upper Quantile of each sample

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miR156 & miR172 are present in Azolla

Nor

mal

ized

Expr

essio

n

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miR156 & miR172 are present in Azolla

𝑟𝑒𝑙𝑒𝑣𝑎𝑛𝑡 𝑒𝑥𝑝𝑟𝑒𝑠𝑠𝑖𝑜𝑛=𝑛𝑜𝑟𝑚𝑎𝑙𝑖𝑧𝑒𝑑𝑞𝑢𝑎𝑛𝑡𝑖𝑡𝑦 𝑜𝑓 𝑚𝑖𝑅𝑁𝐴

𝐴𝑣𝑒𝑟𝑎𝑔𝑒𝑞𝑢𝑎𝑛𝑡𝑖𝑡𝑦 𝑜𝑓 𝑚𝑖𝑅𝑁𝐴(𝑎𝑙𝑙 𝑠𝑎𝑚𝑝𝑙𝑒𝑠)

Rele

vant

Exp

ress

ion

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miR156 & miR172 are present in Azolla

𝑟𝑒𝑙𝑒𝑣𝑎𝑛𝑡 𝑒𝑥𝑝𝑟𝑒𝑠𝑠𝑖𝑜𝑛=𝑛𝑜𝑟𝑚𝑎𝑙𝑖𝑧𝑒𝑑𝑞𝑢𝑎𝑛𝑡𝑖𝑡𝑦 𝑜𝑓 𝑚𝑖𝑅𝑁𝐴

𝐴𝑣𝑒𝑟𝑎𝑔𝑒𝑞𝑢𝑎𝑛𝑡𝑖𝑡𝑦 𝑜𝑓 𝑚𝑖𝑅𝑁𝐴(𝑎𝑙𝑙 𝑠𝑎𝑚𝑝𝑙𝑒𝑠)

Rele

vant

Exp

ress

ion

miR159 downregulates MYB causing sterilitymiR319 reduces TCP protein causing late floweringmiR529 same family as 156

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miR156 & miR172 are present in Azolla

ὁ ἀνεξέταστος βίος οὐ βιωτὸς ἀνθρώπῳ

The unexamined life is not worth living

Σωκρατης απολογία

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miR156 & miR172 are present in Azolla

Acknowledgement

Dr Henriette SchlupmannDr Sayed TabatabaeiLaura DickhousePaul BrouwerValerie BuijsEvelien StoutenJolanda Schuurmans

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To validate the relative enrichment of miRNA in small RNA preparations, quantitative reverse-PCR was carried out using miRNA156 as the target.

smallRNA libraries

miRNA 156 Ct meanFR1 sRNA 21.36FR2 sRNA 25.23TL1 sRNA 21.99TL2 sRNA 25.98FR total RNA 34.17TL total RNA 33.39

Comparison of small RNA and total RNA with exact same amounts of DNA input.

Therefore we proceeded with the small RNA extraction protocol for miRNA sequencing.