Genomics of Ferns and Lycophytes. Chapter 6: Structure and evolution of fern plastid genomes Paul G. Wolf and Jessie M. Roper

Genomics of Ferns and Lycophytes

Chapter 6: Structure and evolution of fern plastid genomes

Paul G. Wolf and Jessie M. Roper

Marchantia cp genome

• ca. 150 kb, circular molecule• large and small single copy regions separated by inverted repeat• gene number and order +/- conserved across land plants

Question:

What is the inheritance of the chloroplast genome in ferns?

Generally in land plants: maternal (via the egg, excluded via sperm)

• maternal with some biparental in Angiosperms• paternal in Gymnosperms• ferns?

Phyllitis (Aspleniaceae) – biparentalOsmunda (Osmundaceae) – maternalPolystichum (Drypoteridaceae) – maternalPteridium Dennstaedtiaceae) – maternalPellaea (Pteridaceae) – maternal

“During insemination in Ceratopteris richardii [Pteridaceae], the sperm cytoskeleton and flagella rearrange, and the coils of the cell extend while entering the neck canal. . . . All cellular components, except plastids, enter the egg cytoplasm”

Lopez-Smith and Renzagalia, 2008 (Sexual Plant Reproduction)

Marchantia cp genome

• ca. 150 kb• large and small single copy regions separated by inverted repeat• gene number and order +/- conserved across land plants

1992

Marchantia

tobacco

30kb inversion

Lycopodium

Equisetum

Psilotum

Osmunda

Lycopodium = Marchantia order

ferns = tobacco order

1992

30kb inversion

Fern and lycophyte total chloroplast genomes sequenced

• Huperzia• Isoetes• Selaginella

• Equisetum (basal fern)• Psilotum (basal fern)• Angiopteris (basal fern)• Adiantum (polypod)• Alsophila (polypod - 2009 paper)*

Gao et al. (2009) Complete chloroplast genome sequence of a tree fern Alsophila spinulosa

Fern and lycophyte total chloroplast genomes sequenced

• few advanced ferns sequenced

• but, Fern Tree of Life project will do many more

Rearrangements in fern chloroplast genomes

1. loss of some tRNA and other protein coding genes Gao et al. 2009


1. loss of some tRNA and other protein coding genes

1. 2 inversions in the Inverted Repeat (IR) of some ferns

Gao et al. 2009


1. loss of some tRNA and other protein coding genes

1. 2 inversions in the Inverted Repeat (IR) of some ferns

30kb inversion

IR inversion 1

IR inversion 2

?

[also using PCR assays for these inversions in other genera]

Chapter 7: Evolution of the nuclear genome of ferns and lycophytes

Takuya Nakazato, Michael S. Barker, Loren H. Rieseberg, and Gerald J. Gastony

Unfurling fern biology in the genomics age (BioScience, 2010)

Michael S. Barker and Paul G. Wolf

Academic family tree of Gerald J. Gastony

Rolla and Alice Tryon 1950s and 1990s

Is there an “Alice Tryon Women in Science” bequest for Botany Department?

Academic family tree of Gerald J. Gastony

Rieseberg

Nakazato

Barker

The neglected fern and lycophyte nuclear genomes

1. 1 genetic linkage map - Ceratopteris

1. 4 EST libraries – Selaginella (2), Ceratopteris, Adiantum

2. 3 BAC libraries - Selaginella (2), Ceratopteris

3. 1 nuclear genome sequencing project in the works - Selaginella

- or the “crying ferns”


Why?


1. large genome size (>2X)

1. lack of funding for low economically important plants


Why?


1. large genome size (>2X)

1. lack of funding for low economically important plants

But !

1. 2nd largest land plant group

2. sister to seed plants

3. diverse land plant lineages need to be compared

4. homologs of important seed plant genes occur in ferns

A short history of the study of the fern genome

Haploid chromosome number

• 57 in ferns vs. 16 in angiosperms [ > 14 = polyploid (Grant, 1981) ]

Ophioglossum (adder’s-tongue fern) - 2n = 1440 (96 ploid) in O. reticulatum




Questions:

How does this fern choreograph meiosis with an n > 600? Has it ever been observed? Do large n's lead to more aborted or nonviable spores?




• 13.6 in heterosporous ferns is exception

• heterosporous lycophytes << homosporous lycophytes

• heterosporous seed plants << homosporous ferns & allies

Therefore, homosporous ferns acquire high chromosome number to select for increased heterozygosity via polyploidy

Hypothesis of Klekowski & Baker (1966)


Therefore, homosporous ferns acquire high chromosome number to select for increased heterozygosity via polyploidy

Hypothesis of Klekowski & Baker (1966)

Two lines of evidence did not support this hypothesis

1. Isozyme analysis indicated widespread silencings of genes – diploid numbers of copies

1. nn2. Most homosporous ferns are

outcrossing


Homosporous ferns acquired high chromosome numbers with diploid gene expression via repeated cycles of polyploidization and subsequent gene silencing without chromosome loss

Hypothesis of Chris Haufler (1987)


Many lines of evidence support this as the working hypothesis in ferns

1. Pseudogenes in nuclear genes in Polystichum

1. FISH detection of multiple dispersed chromosomal locations of rDNA in Ceratopteris

1. +/- Genetic linkage map analysis in Ceratopteris

Homosporous ferns acquired high chromosome numbers with diploid gene expression via repeated cycles of polyploidization and subsequent gene silencing without chromosome loss

Hypothesis of Chris Haufler (1987)

The future of fern genomics?

Ceratopteris has emerged as the “model” organism for fern genomics

Study of the origin of polyploidy (neo- and paleo-)

Correlating genomic changes to speciation and development

Two examples using Ceratopteris

1. Nakazato et al. (2006) genetic linkage analysis

1. Barker (2010) EST analysis


Ceratopteris genetic linkage analysis

• 700 genetic markers

• 85% multiple copies

• 24% single copy – low!

• large numbers of duplicate genes on different chromosomes


Ceratopteris genetic linkage analysis surprises!

• Expect clusters of linked duplicate genes on different chromosomes in recent (neo-) polyploids

Maize linkage map

Oxford plot of polyploid cotton’s A & D genomes

Rong et al. 2004

• Expect clusters of linked duplicate genes on different chromosomes in recent (neo-) polyploids

Duplicated gene copies are hyper-dispersed across the genome of Ceratopteris

• Expect clusters of linked duplicate genes on different chromosomes in recent polyploids

Indicates ancient polyploid event and many subsequent chromosomal changes


Ceratopteris EST analysis

• expressed sequence tags

• examines transcriptome

• mRNA is extracted



• cDNA is made with reverse transcriptase

• ds cDNA is cloned into vector – library formed

• cDNA sequenced from 5’ and 3’ ends (= Tags)

• 400-800 bp ESTs can be contiged



• synonymous substitution (silent) rate – Ks – obtained for duplicate genes

• most duplications young and placed in ‘zero’ class

• peak in duplications at 0.96 – 1.84 Ks or showing paleopolyploidy



• synonymous substitution (silent) rate – Ks – obtained for duplicate genes

• most duplications young and placed in ‘zero’ class

• peak in duplications at 0.96 – 1.84 Ks or showing paleopolyploidy

• using molecular clocks and phylogenetic trees, paleopolyploidy linked to early polypod diversification

Question Set 1

1. Ferns and fern allies are diverse and old; is it really appropriate to expect that all have their nuclear genomes evolving by same “rules”?

1. You have been given a blank check to sequence the fern genome of yourchoice. Which would you choose and why? What methods would you use?

2. Why is the fate of most duplicate genes to eventually become silenced? Could mutations accumulate in both copies at the same rate causing subfunctionalization, where mutations cause the two copies to functionally be diminished to one over time?

3. If you are really interested in understanding the process of speciation, would ferns be the better choice relative to angiosperms?

1. What are the justifications for selecting Ceratopteris richardii as a model organism for ferns? Do the “idiosyncratic” features of its genome affect generalization to ferns?

2. Could maintaining large amounts of physical genetic material be disadvantageous for fern evolution? Could it be related to slow speciation rates, compared to angiosperms? Or, on the other hand, could the silenced genes hold the key to the long history of fern evolution?

1. Can high chromosome numbers in ferns and lycophytes simply be an outcome of the ‘stringent bivalent pairing’ that is known in the group? How might that idea be further examined or tested?

Question Set 2

Documents

Genomics of Ferns and Lycophytes. Chapter 6: Structure and evolution of fern plastid genomes Paul G. Wolf and Jessie M. Roper