Plant molecular genetics
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Plant genome
Chromatine and DNA methylation
RNA interference
Genome of plastids and mitochondria
Transposible elements
Viruses
Classical genetic mapping
Transgenosis and reverse genetics
Genomics, next generation sequencing
Transcriptomics
Proteomics
Components of plant genome
• nuclear genome = genome sensu stricto
• plastids - plastome
• mitochondria - chondriome
Plant genome sizes
54 Mbp – Cardamine amara
124 852 Mbp - Fritillaria
149 000 Mbp - Paris japonica
- currently the largest
(not only plant)
http://data.kew.org/cvalues/
Plant genome sizes
10 Mb Ostreococcus (single cell alga)
54 Mb Cardamine amara
64 Mb Genlisea aurea
Ratio of globe volumes differing 3000 times
125 Mb Arabidopsis
500 Mb Oryza
5 000 Mb Hordeum
17 000 Mb Triticum
84 000 Mb Fritillaria (largest diploid)
143 000 Mb Paris (oktaploid)
- Angiosperms – size differences up to almost 3 000 times
- Gymnosperms – genome sizes often around 10 000 Mb
- Gene number differences much lower (approx. 20 – 200 fold)
Plant genome sizes
What we can deduce?
- Genomes are increasing in
evolution
- Average increase is higher
in Monocots
C-value paradox
- there is no strong correlation between complexity
of an organism and the size of its genome
• C-value = size of genome in non-replicated gamete
genome size (bp) = (0.910 x 109) x DNA content (pg)
DNA content (pg) = genome size (bp) / (0.910 x 109)
1 pg = cca 910 Mbp; MW (1 bp) = cca 660 Da
• genomes of related organisms often strongly differ in size
causes:
- duplications of whole genomes (polyploidization) or
chromosome segments
- replication of invasive DNA (transposable elements)
- but reductions also possible (recombination – diploid cotton sp.)
Sequences in plant genomes
Unique sequences – genes, but also non-coding (!)
Repetitive:
• Duplications of chromosomal regions
• Medium repetitive DNA
– Tandem repeats of rRNA, tRNA a histon genes
– Gene families with multiple members
– Transposable elements – also high repetitive
• Highly repetitive – low complexity DNA
- Tandem arranged simple sequence repeats (SSR)
– Centromers (180 bp repeat Arabidopsis) a telomers
(TTTAGGG)n
Types of sequences in plant genomes
• Unique sequences – coding genes, but also noncoding regulatory (!)
• Medium repetitive DNA
– Tandem repeats of rRNA, tRNA a histon genes
– Gene families with multiple members
– Transposable elements – also highly repetitive
• Low complexity DNA (highly repetitive)
– Tandem arranged simple sequence repeats (SSR)
– Centromers (180 bp repeat Arabidopsis) a telomers
(TTTAGGG)n
- some behave as satelite DNA
Aside – term definition:
sequence complexity
(~ the amount of information)
repetitive
AAAAAAAAAAAAAAAAAAAAA complexity 1 (21xA)
ATCATCATCATCATCATCATC
complexity 3 (7xATC)
(what is the complexity if it is a coding sequence?)
unique
ATCGTATCGCGATTTTAACGT
complexity 21 (1xAT…)
- unique x repetitive – depends on the size of the evaluated frame
(= size of analyzed DNA fragments)
Sequence complexity of plant genomes
Higly repetitive
Medium repetitive
Unique
Sequence
complexity
Examples of repetitive DNA representation
in u Soybean and Silene (clusters of related sequences)
Silene latifolia
Gypsy, copia
= retrotransposon families
clDNA
= chloroplast DNA
(partially contamination, but
also recent insertions)
Measuring of genome complexity reasociation kinetics
• DNA fragmented to 300 - 500 bp, denatured
• Monitoring of reassociation in time - separation
(chromatographic) of ss and ds DNA
• Analysis of kinetics (Cot curves) shows
representation of various types of repetitive DNA
– rare sequences reasociate more slowly that
repetitive
Reasociation kinetics
depends on sequence complexity
Eucaryotic genomes usually contain three
fractions of sequences with different
complexity
Low complexity = highly repetitive
Middle repetitive
Unique sequences =
High complexity
Reasociation kinetics of small and large genomes
Unique
Medium repetitive
Highly repetitive
Repetitive sequences can be easily detected in situ
FISH = fluorescent in situ hybridization (possible even with unique seq.)
180 bp A.th.
45S rDNA Crocus
copia A.th.
tandem repeats dp5a1 wheat
(Heslop-Harrison, Plant Cell 12:617, 2000)
Subtelomeric repeats in rye
(Heslop-Harrison, Plant Cell 12:617, 2000)
Telomers in rye
(TTTAGGG)n
Differences in small and large genome arrangements
large genomes: genes present in „gene-rich islands“
isolated with long regions of repetitive DNA
Reconstruction of gradual cummulation of transposable
elements in maize genome
In Panicum in the presented region no transposible elements, in maize 60 % of its size
Plant Genome Sequencing
http://genomevolution.org/wiki/index.php/Sequenced_plant_genomes
April 13 – less complete in gray
Large Genome Sequencing
- sequencing per partes (separated chromosomes)
- sequencing of non-methylated DNA
(= transcriptionally active)
- sequencing of ESTs
Aside – term definition:
Expressed Sequence Tags (ESTs)
- short sequenced regions of cDNA (300-600 nt)
- mostly gene segments (primarily from mRNA)
- alternative sourse of coding sequences for large genomes
(rapid and inexpensive)
Weak points:
- highly redundant, incomplete (!)
- problems: various transcript levels
- gene expression regulated spatially and
temporally, developmentally, environmentally
- regulatory sequences not represented
(promotors, introns,...)
Expressed Sequence Tags (ESTs)
Preparation of EST library
- mRNA
- RT with oligoT primer  cDNA
-cleavage of RNA from heteroduplex
RNAseH
- 2nd strand cDNA synthesis
- cleavage with restriction endonuclease
- adaptor ligation
cloning
sequencing
Aside:
Arabidopsis
thaliana
1 week
3 weeks
the most important
model of plant
biology
4 weeks
6 weeks
Arabidopsis genome: 125 Mbp
genes
ESTs
TEs
genes
ESTs
TEs
genes
ESTs
TEs
genes
ESTs
TEs
genes
ESTs
TEs
High density
low density
Total gene number prediction in time
(after whole genome sequencing)
Genome of Arabidopsis statistics
Value
Feature
DNA molecule
Chr.1
Length (bp)
Top arm (bp)
Bottom arm (bp)
29,105,111
14,449,213
14,655,898
Base composition (%GC)
Overall
Coding
Non-coding
Number of genes
Gene density
(kb per gene )
Average gene
Length (bp)
Average peptide
Length (bp)
Exons
Number
Total length (bp)
Average per gene
Average size (bp)
Number of genes
With ESTs (%)
Number of ESTs
Chr.2
Chr.3
Chr.4
Chr.5
SUM
19,646,945
3,607,091
16,039,854
23,172,617
13,590,268
9,582,349
17,549,867
3,052,108
14,497,759
25,53,409
11,132,192
14,803,217
115,409,949
33.4
44.0
32.4
35.5
44.0
32.9
35.4
44.3
33.0
35.5
44.1
32.8
34.5
44.1
32.5
6,543
4.0
4,036
4.9
5,220
4.5
3,825
4.6
5,874
4.4
2,078
1,949
1,925
2,138
1,974
446
421
424
448
429
35,482
8,772,559
5.4
247
19,631
5,100,288
4.9
259
26,570
6,654,507
5.1
250
20,073
5,150,883
5.2
256
31,226
7,571,013
5.3
242
60.8
56.9
59.8
61.4
61.4
30,522
14,989
20,732
16,605
22,885
25,498
132,982
33,249,250
105,773
+ hundreds of MIR genes - role in regulation of gene expression
Gene function
The majority of plant genes form
gene families
Number of paraloques
• gene families are often in tandem arrangement, but also spead in the genome
• tandem repeats are composed of near, but also far paralogues (recombinations)
• duplications of long chromosomal regions
Aside – terms definition:
Homologous genes
genes with similar sequences derived from the same ancestral gene
(quantification – sequence identity, similarity)
• Paralogous genes
genes with similar sequences derived from the same ancestral gene present
at different loci within the same genome.
• Orthologous genes
genes in different species that are similar to each other because they originated
from a common ancestral gene in a common ancestor.
(if more paralogues are present – genes serving the same function are
regarded to be orthologs)
Orthologues vs. paralogues
Orthologous genes
Species A
Gene A”
Ancestral
Gene A
Species
Species B Gene A’
Paralogous genes = genes duplicated within the species
Species A
Ancestral
Gene A
Species
Gene A” Gene A’”
Paralogous genes
Species B Gene A’
Mechanisms of gene
duplications
(increase in paralogue number)
• tandem duplication
• transpozition
• segmental duplications
• whole genome duplications
Differences in genes/gene families in genomes
Genes
Gene families
Arabidopsis x Populus – large overlap, about 1,5 times more paralogues in poplar
(Arabidopsis + Populus) x Oryza – many genes specific for Monocots
Arabidopsis is ancient tetraploid
(as well as probably the majority of plants)
Duplicated chromosomal regions form about 60 % of genome (67.9 Mb)
Polyploidization significantly increases genome (and organism) plasticity
and played very important role in plant (genome) evolution;
About 30-80% plant species are polyploid
Polyploidization in Angiosperm evolution
Fawcett et al. 2009
Dating of whole genome duplication
according to the number of synonymous mutations per synonymous site - Ks
Ks=3/2,66
Phe Leu Met Val
UUU CUA AUG GUU
UUC UUG AUG GUU
0 0 1/3 1/3 0 1 0 0 0 0 0 1
Gene number
= number of syn. sites
Comparisons of paralogue pairs
Peaks indicate genome duplications
Ks
Fawcet et al. 2013
Polyploidization in plant evolution
• 35 % species neopolyploids
• most species repeatedly polyploid in evolution
• viable aneuploid variants –
(frequetly after allopolyploidization – hexaploid wheat)
stabile wheat lines with missing chromosomal arm (of homeologic
chromosome)
Blue dots – duplications,
asterix – triplication
K-T
(Fawcett et al. 2013)
Polyploidization
- fusion of non-reduced gametes or endoreduplication
n=x=4
n=x=4
n=x=4
x
x
2n = 4x = 16
n=x=7
Spontaneous duplication (endoreduplication)
2n = 4x = 22
autopolyploidy
allopolyploidy
Similar frequency in polyploidic plant species
Chromosome doubling is necessary for
meiosis in hybrids
species A
species B
X
sterile
Genome
duplication
fertile
Preferential pairing of homologous chromosomes
Related from different species (homeologous) can
also pair
Allopolyploidic genomes in Brassica genus
BB
Species
Caryotype Genom
e
Brassica
rapa
2n = 2x =
20
A
B. nigra
2n = 2x =
16
B
B.
oleracea
2n = 2x =
18
C
B. juncea
2n = 4x =
36
AB
B. napus
2n = 4x =
38
AC
B. carinata 2n = 4x =
34
BC
Brassica nigra
BBCC
AABB
Brassica juncea
Brassica carinata
CC
Brassica olarecea
Ancient
interspecies
hybrids
AACC
Brassica napus
AA
Brassica rapa
Allopolyploid tobacco species – DNA size changes
Fade of duplicated genes differ
(gene dosage balance theory)
genes encoding interacting proteins “connected genes“
(signal pathways, complex subunits, …) easily preserve in
genome after duplication
•
- loss or partial duplication of one component results in gene
inbalance decreasing fitness,
- whole duplicated complex can be specialized for a new
function and increase organism complexity
-secondary function probably present already in the
ancestral complex (pathway), but only duplication allowed
adaptive evolution for both functions without selection
constrains - Escape from adaptive conflict - EAC model
•
other „single genes“ more easily lost after genome duplication,
but can be preserved after individual duplication
- most of duplicated genes is lost after whole genome duplication
- loss is not as even (↑) in both copies
- probably frequent epigenetic marks in one copy (methylation)
- preferential gene loss and mutagenesis of methylated copy
- gene conversion and homogenization can occur (!)
de novo allopolyploids (~ rape seed) – recombinations preferentially in
homeologous chromosomes without preference of any parental genome
(= homologní, v jednom genomu, ale původem od různých rodičů)
Changes in newly formed allopolyploid genome:
- DNA methylation changes
- losses of parts or whole chromosomes (aneuploidy
– decreased fertility)
- frequent activation of TE
- expression of homeologous genes is not usually additive
- transcriptome usually more reduced than genome
- different regulation of expression - often organ specific
expression of genes from
each parent, new sites
of expression, new regulation
- „divergent resolution“ - speciation
(different gene loss in individuals - lethality in F2,
- absence of essential gene = reproduction barrier
Plants can survive also with haploi genome!
- reprogramming of male or female gametophyte development in vitro – no gamete
formation, but development resembling embryogenesis
- usually from immature microspores = androgenesis
- female gametophyte = gynogenesis
- haploid plants are sterile
- through endoreduplication (colchicin or spontaneous) – completely
homozygous plants – dihaploids
Androgenesis in rape seed (pollen embryogenesis)
... But genomes are still similar
Colinearity, syntheny
Paterson et al., Plant Cell 12: 1523-1539, 2000
„Syntheny“ is usually missused to describe
colinearity
Syntheny = orthologous loci in two species on the same chromosome
Species A
Ancestral
Species
A
B
C
Species B
A’
C’
B’
C”
B”
A”
Colinearity = group of loci in two species on a chromosom in the same order
Species A
Ancestral
Species
A
B
C
Species B
A’
B’
C’
A”
B”
C”
Changes in colinearity caused by
chromosomal arm inversion
Colinearity of Poaceae genomes
Colinear regions differ mainly in repetitive DNA
Summary:
• Current plant genomes result from
repeated cycles of partial and complete
duplications, followed by reduction and
modification of duplicated sequences.
• There are no genomes without
redundancy.
• Plant genomes are still very dynamic.
• High portion of genome consists of
repetitive DNA