conservation of plant genetic resources.

CHALLENGES IN RESEARCH AND

CONSERVATION OF TROPICAL PLANTS:

Symphonia as an example

20.4.2016 by Sanna Olsson

[email protected]

Malagasy congeners

Symphonia

globulifera

The genus Symphonia (Clusiaceae)

• Symphonia has radiated on Madagascar (16-23 endemic

species) (Perrier de la Bâthie 1953, Abdul-Salim 2002)

• One widespread species: Symphonia globulifera L. f.

Symphonia

fasciculata

(Noronha ex

Thouars) Vesque

Symphonia L.f.,

Specimen voucher:

Lowry et al. 4209

Symphonia louveli

Jum. & H. Perrier

ITS phylogenetic analysis, Dick et al. 2003, Am Nat

CHALLENGES IN RESEARCH AND

CONSERVATION OF TROPICAL PLANTS

• Difficult access to sampling sites - A lot of bureaucracy involved

- Lack of infrastructure for handling material

• Unkown taxonomy - Species concepts based on morphology only

- Overlapping concepts on different continents

• Lack of genetic reference data - Lack of funding at local institutions

- Secondary metabolites complicate molecular work

- Model species vs. non-model species

Bureaucratic procedures for sampling permissions

• Start planning several months earlier

• All administrative levels: from Customs to villages

• Local help valuable

• Knowledge of local language important

Bureacratic procedures for sampling permissions

Unkown taxonomy

• Incomplete taxonomic

information

• Scarcity of good herbarium

vouchers

• Poor knowledge on

distribution

Lack of genetic reference data

• Funding needed for molecular work

• Difficulty of obtaining fresh material

• Secondary metabolites commonly present

Applications of PCR-amplification

• Genotyping

- Sequence variation between individuals

- Population structure

- Genomic- or transcriptomic markers (SSR and SNP)

• Phylogenetics

- Sequence differencies between species

• Gene function

- Mutant vs. wildtype

• Repeating sequences of polymorphic DNA loci

• Reference genome greatly facilitate marker development

SSR = Microsatellite markers

SSR = Microsatellite markers

>IR_3591 ??????????????????????????????????

??????????????????????????????????

??????????????????????????????????

??????????ATTCGCGATTACGCTGAGGATGGC

GATGACGACGATGATGATGGAGAGGACGAGGATG

AGGATGAGGACGAGGATGAGGAAACGATGACGAT

GATGATGATGATGATGATGATGACGACGAAGTCG

ACATCACCAAGCGTGCTACCCT

Phylogenetic markers

Chloroplast • rpl16

• trnL-trnF

Nucleus

• nuclear ribosomal ITS 1+2

Mitochondria

• nad5 intron

http://de.wikipedia.org/wiki/Bild:Di agram_human_cell_nucleus.svg

http://chloroplast.cbio.psu.edu/ http://en.wikipedia.org/wiki/Mitochondrion

Phylogenetic analysis

• Same markers regions used

• Include non-coding regions

• Single point mutations and indels

SNP detection

• Synonymous vs. non-synonymous changes

• Study of genetic function requires good annotations

(reference genome)

• Useful for phylogenies

Actin mutant wavy in Arabidopsis

Arg  Cys

Symphonia sampling in Madagascar (2010 & 2013)

• Establishment of 2 sympatric

plots

• Masoala National Park: expected

Symphonia verrucosa, S. urophylla

and possibly S. pauciflora or S.

sessiliflora

• Andasibe-Mantadia National Park:

expected Symphonia pauciflora, S.

urophylla and possibly S. verrucosa

or S. louvelii

Andasibe-Mantadia

Nosy Mangabe

Symphonia in Madagascar: sympatric

species common, species complexes?

Symphonia cf. urophylla in valley bottoms Symphonia cf. louvelii

in midslopes

Symphonia

sp. 3 (?)

Segregation by habitat in the Andasibe Mantadia

National Park

• The easily identified

Symphonia cf.

urophylla has 6

ribotypes

• The morphologically

diverse S. cf. louveli

and putative sp. 3

have R12 and R14

300 m

Population genetic structure in Andasibe

psbS-trnA Bayesian phylogenetic tree

(preliminary): bad resolution

Symphonia spp. from Madagascar nested within S. globulifera

• No shared ITS ribotypes between North and Central Madagascar, despite 3 shared species expected

• psbA-trnH indicates plastid capture

ITS and psbA ineffective for species delimitation

Symphonia

sampling in

Madagascar

2010, 2013,

2014

Symphonia microphylla Benth. et Hook. f. ex. Vesque,

individual MH3026

Symphonia cf. eugenioides, individual MH3020

Symphonia cf.

eugenioides,

individual

MH3082

Reference genomes

• Ideal situation vs. real life

• Alternative splicing & repetitive regions

• Well-annotated de novo genome laborious

• Fragmented draft genomes (non-model species)

• Shallow coverage, sequence errors

• Helpful in marker development!

Sex determination in papaya by gel electrophoresis

Deputyet al. 2002:Molecular markers for sex determination in papaya

Material preparation for RNAseq

• Fresh high-quality material

• Controlled environmental / experimental conditions

• From different tissue types

• Avoid degradation by RNAse (RNAlater)

 Difficult to fulfill in tropical field conditions!

RNAseq applications

• Gene expression differences between:

- wild type and mutant

- developmental stages

- populations

- sexes

 Very few examples in tropical species

• Useful for marker developement (across species)

Secondary metabolites

RNA extraction from Symphonia

• Problems with secondary metabolites

• Qiagen RNeasy

• Tri reagent

• Phenol extraction by Ghawana et al. 2011: An RNA

isolation system for plant tissues rich in secondary

metabolites

• Norgen

• RIN > 8 in 11 samples, 14-123 ng/µl

Strategy to solve evolutionary relationships in

Malagasy Symphonia

• Herbarium material for morphological identification

• Microsatellite markers to solve genetic clusters using our

samples and herbarium vouchers if possible

• Transcriptomics – (partially) solve phylogeny, identify

signals of adaptation, basis for capture experiment

• Gene capture in 96 or 192 individuals chosen on the basis

of SSR analysis

CONCLUSIONS

• Established collaboration with local researchers

• Optimize the use of rare, valuable DNA samples!

• Genomic DNA can be amplified with whole genome

amplification (WGA) to increase quantity

• High-throughput sequencing for tropical taxa:

whole-genome sequencing and transcriptomics