Molecular markers used plant phylogeny

Introduction

Well-resolved phylogenetic trees are essential for understanding evolutionary processes.…

Higher level - Protein sequences / coding regions Lower level - Non-coding regions: introns and intergenic regions

Nuclear regions – paralogs, concerted evolution etc – more informative – species level – subspecies level

Plastid regions – single copy maternally inherited – family/generic level

Mitochondrial regions – low sequence variation

Different genomes – different markers

Internal transcribed spacer (ITS)

White et al., 1990

Universal primers are available – White et al., 1990

High copy number - herbarium material too

Small size ~700 bp

High variation to study at species level

Can detect hybridization, ploidy etc.,

External transcribed region (ETS)

Calonje et al., 2009

ncpGS - introns

Nuclear encoded chloroplast expressed glutamine synthaseOxalidaceae – ncpGS / ITS – (Emshwiller and Doyle 1999 )Diverged much earlier from cytosolic GS – single copy in most taxa.Widely used eg., Gesneriaceae, Arecaceae, Apocynaceae…. Etc

Universal primer

Can be used along with other cpDNA markers to test congruence of data

Other introns:PEPC – fourth intronadh – introns between exons 2 and 9

AP3/DEF

MADS box transcription factor – B function gene

Impatiens – inter species levelTwo copies – paralogsPrimers have to be developed per group

Other MADs box genes used FLO/FLY – 2nd intronPI – 1st intron , 4th and 5th intron etc ..

Microsatellite (SSRs) flanking regionsSSRs have long been used in phylogeny and population studies.Flanking regions of SSRs

Annona - LMCH9/10Neutral evolutionMuch more variable - short length

Chatrou et al 2009

Transposable elements

Used to study populations along with AFLP Widely used in DNA fingerprinting than in phylogeny reconstructions at supraspecific level

Aid of Genomics

Next-generation sequencing – 454 sequencing – Illumina.

Genome assembly – provides ample information for marker development.

Rapid and cheaper sequencing of multiple regions across organisms (multiplexing)

0.5 x genome sequencing

complete chloroplast genome of 158,598

complete rDNA cistron (18S-5.8S-26S; 7,541 bp) and 5S rDNA (120 bp)

partial mitochondrial genome sequence (130,764 bp)

88% hit with Cartharanthus roseus Unigene data

66% hit with orthologs of asterids

- Population level as well as species level microsatellite markers were developed

Microsatellite marker development using genomic information

> 150 bp contigs were scanned for 2,3,4,5,6 bp repeats - with Batchprimer3inverted repeats and several flanking – 100 and 450 bp

Genomic approaches

NGS methods - RAD sequences – Illumina UCE – Ultra conserved elements

Targeted amplicon sequencing – Roche 454 Multiplexing using MIDs or Barcodes – 3 cp and 2 nuclear

Assembly of reads > Quality check > Alignment – Phylogeny

Population level studies are possible – Haplotype networks

Presence of multiple alleles can be detected without cloning in polyploid species

Griffin et al., BMC Biology 2011, 9:19

Target Enrichment is the first step in genome wide sequencing

Hybridization based enrichment methods (Sequence capture) - can nowadays replace PCR as a method of enrichment

One or more genomes or transcriptomes are necessary for designing hybridization probes for sequence capture

90 base pair - synthesized probes would be used to capture sheared

DNA library prepared from target species using a Biotin-Streptavidin bead based binding.

The targeted regions will be harvested and sequenced as short reads by paired end sequencing using Illumina HiSeq NGS platform

Mitochondrial DNA

In higher plants: 4 times slower than chloroplast DNA (cpDNA) 100 times slower than in animal mtDNA

The genome size is varied in different groups rate of rearrangements is extraordinarily faster in plant mtDNA than in cpDNA and animal mtDNA

Used in phylogenies at lower groups of plants

Palmer and Hebron, 1998

Chloroplast DNA

Single copy number – no paralogy

Inherited from single parent

Lots of copies in cell

Widely used in plant phylogenetics

●Coding regions●Introns ●Intergenic spacers

rbcL

ribulose-1-5-biphosphate carboxylase/oxygenase (rbcL)Ritland and Clegg (1987)

Widely used for family level or higher classification

APG II system :18S rDNA, rbcL, and atpB

matK

maturase K – about 1.5 kb – one of the fast evolving genes

Embedded in between the trnK intron

429 and 1313 of the matK (~930) – universal primer

Potential candidate for plant DNA barcode along with other regions

trnTL-trnL-trnLF

The Tortoise and the Hare• Screening for newer non-coding cpDNA regions

– 34 noncoding regions – from different groups of APGII

• Primers based on 3 complete genomes – Poaceae, Fabaceae and Solanaceae. (Shaw et al. 2005, 2007)

Combined regions●trnT-trnL-trnL, ●ndhJ-trnF-trnL, ●TrnS-trnG-trnG, ●2 trnK intron, ● trnL-trnLtrnF.

DNA barcoding

• www.barcodinglife.org - CBOL• http://barcoding.si.edu/• http://www.dnabarcoding.ca• http:// www.kew.org/barcoding• http://www.ibolproject.org.

• matK, rpoC1, rpoB, accD, YCF5 and ndhJ : CBOL (2008)

• rbcL + matK : CBOL (promising candidates)

Roy et al ., 2011. PLOS one

psbA-trnH, matK, rbcL & ITS

Berberis, Ficus, Gossypium

Problems with plant barcodes

• ITS has multiple copies (paralogs) in several taxa

• psbA-trnH is rapidly evolving and has lot of indels

• psbA-trnH and ITS were not able to differentiate species of Scalesia (Asteraceae). (Seberg and Peterson ., 2010)