Leibniz Institute of Plant Genetics & Crop Plant Research (IPK), Gatersleben, Germany
Chromosomal integration of the
Spirodela polyrhiza reference genome
Hieu Cao
the 2nd Inter. Conference on Duckweed Research & Applications
New Brunswick, Aug 21-24, 2013
Lemnoideae (duckweeds) o 5 genera, 37 species; worldwide distribution, except
arctic & antarctic zones
o Floating free on surface water; neotenous reduction
o Fast growing & rare sexual reproduction
o Genome size variation: 0.158 – 1.88 Gbp
o Chromosome numbers (2n): 20 - 126
Drawings: Dr. K. Sowjanya Sree, Amity University Delhi.
2 mm 10 mm
Spirodela
1 mm
Wolfiella
1 mm
Wolffia
Lemna 5 mm
Landoltia
Lemnoideae are of interest for genome
and karyotype evolution studies
Wolfiella
Lemna
Landoltia
Spirodela
Wolffia
< 0
.25
0.2
5 –
0.5
0.5
- 1
1 –
1.5
> 1
.5
*
*
Les et. al. Sys. Bot. 2002 Wang et. al. J. Bot. 2011
Geber Uni. Vienna 1989
Landolt VGI-ETH 1986
(*) Most common value within the genus
Appenroth et al. CIBJ 2013
low
high
Pla
stid D
NA
substitu
tion r
ate
s
Nauheimer et. al. N. Phytol. 2012
2n = 42* [20 – 62]
2n = 42* [20 – 50]
2n = 42* [20 – 126]
2n = 46* [40 – 50]
2n = 36/40*[20 - 80]
DNA content
pg / 1C
Preliminary cytogenetic characterization
of Spirodela polyrhiza clone 7498
1 2 3
4 5
6
7 8 9
10
11
12
13 14
15
16 17
18
19
20
21 22 23
24 25
26
27
28
29
30
31
32
33
34 35
36 37
38
39
40
2n = 40
10 µm
S. polyrhiza clone 7498 contains 20 chromosome pairs (somatic metaphase)
1 2 3
4 5
6
7 8 9
10
11
12
13 14
15
16 17
18
19
20
21 22 23
24 25
26
27
28
29
30
31
32
33
34 35
36 37
38
39
40
2n = 40
Preliminary cytogenetic characterization
of Spirodela polyrhiza 7498
Genlisea filiformis (179Mbp) Spirodela polyrhiza 7498 (158Mbp)
H3K4me2 DAPI
PI 5mC
DAPI H3K4me2
PI 5mC
Typical heterochromatic chromocenters
of small genomes are free of H3K4me2
but enriched in 5mC.
Eu
ch
rom
atin
ma
rk
He
tero
ch
rom
atin
ma
rk
Constitutive heterochromatin which is
visualized in S. polyrhiza interphase
nuclei by propidium iodide (PI) staining
is enriched in 5mC.
“As more genomes are assembled from
scratch, scientists are struggling to assess
and improve their quality”
FISH as a finishing genome tool
o Challenges: Genome size, complexity (heterozygosity and/or ploidy),
repetitive sequence content and composition
o Integration of multiple sequencing technologies, mapping and transcriptomics
datasets
o Finishing genome: gap closure, assembly validation and refinement
o Multicolor Fluorescence in situ Hybridization (mcFISH): o to map genomic fragments/ library clones to specific chromosomal regions
o to validate the integrity of the WGS scaffolds
o to validate and support the integration of WGS and physical maps
o to detect chromosome rearrangements, chromosome homeology and karyotype evolution in
related non-sequenced species
o Spirodela polyrhiza / duckweeds contains: o high number of small chromosomes
o no/very difficult to obtain genetic maps
Strategy for chromosomal integration
of pseudomolecules (Ψs) by FISH
WGS assembly scaffolds
Fingerprinting
maps
32 pseudomolecules
96 presentative BACs
96 presentative BACs
Test for the integrity of Ψ
by differently labelled BACs from each Ψ
Chromosomal integration of Ψ
by differently labelled BACs from pools of Ψs
Genomic and Genetic data Cytogenetic integration
Validation of the integrity of
the pseudomolecules
023I01 034P03
035L19 002I04
023I01 034P03
035L19 002I04
Ψ6 (6.3 Mbp)
023I01 034P03 002I04 035L19
Ψ6 is present on one chromosome pair
Validation of the integrity of
the pseudomolecules Ψ7 (6.2 Mbp)
006B02 004N06 002B12 035P14 009O07
006B02 004N06 002B12 035P14 009O07
006B02 009O07 004N06 002B12
035P14 merged merged 1st FISH
006B02 009O07
004N06 035P14 002B12
merged merged 2nd FISH
Ψ7 is split into two chromosome pairs
Results of chromosomal integration
of pseudomolecules (Ψ) by FISH
WGS assembly scaffolds
Fingerprinting
maps
32 pseudomolecules
96 presentative BACs
96 presentative BACs
Integrity of 30 Ψs is confirmed
2 chimeric Ψs are detected
32 +2 Ψs are intergrated into 20
chromosome pairs
Genomic and Genetic data Cytogenetic integration
Integration of 32 + 2 Ψs into
20 chromosomes
Ψ24,25,26 Ψ30,31,32 Ψ27,28,29 1st FISH
Ψ25 & 28 belong one chromosome pair
Ψ25 Ψ24 & 28 Ψ27
25_28
25_28
27
27
24
24
2nd FISH
8.9 Mbp
8.8 Mbp
11.4 Mbp
8.5 Mbp
6.5 Mbp
8.1 Mbp
6.9 Mbp
7.7 Mbp
5.5 Mbp
8.1 Mbp
4.7 Mbp
7.2 Mbp
6.4 Mbp
4.6 Mbp
8.3 Mbp
4.4 Mbp
5 Mbp
3.5 Mbp
3.4 Mbp
6.7 Mbp
Largest size: 11.4 Mbp
Smallest size: 3.4 Mbp
Average size: 6.73 Mbp
Total size: 134.6 Mbp
34 Ψs
Integration of 32 + 2 Ψs into
20 chromosomes
8.9 Mbp
8.8 Mbp
11.4 Mbp
8.5 Mbp
6.5 Mbp
8.1 Mbp
6.9 Mbp
7.7 Mbp
5.5 Mbp
8.1 Mbp
4.7 Mbp
7.2 Mbp
6.4 Mbp
4.6 Mbp
8.3 Mbp
4.4 Mbp
5 Mbp
3.5 Mbp
3.4 Mbp
6.7 Mbp
4 chromosomal linkages flanked by 2 telomere repeat termini
20 predicted telomere repeats 34 Ψs
Telomere repeats
12 chromosomal linkages flanked by 1 telomere repeat terminus
Integration of 32 + 2 Ψs into
20 chromosomes
8.9 Mbp
8.8 Mbp
11.4 Mbp
8.5 Mbp
6.5 Mbp
8.1 Mbp
6.9 Mbp
7.7 Mbp
5.5 Mbp
8.1 Mbp
4.7 Mbp
7.2 Mbp
6.4 Mbp
4.6 Mbp
8.3 Mbp
4.4 Mbp
5 Mbp
3.5 Mbp
3.4 Mbp
6.7 Mbp
34 Ψ
Spirodela cytogenetic map
Largest number of markers per chromosome: 8
Smallest number of markers per chromosome: 2
Average number of markers per chromosome: 4
Total number of markers: 85
85 BAC markers
Future tasks
o Cytogenetic characterization of further Lemnoideae species
o Introducing more BAC markers in order to (1) define the break points of
2 chimeric Ψs; (2) determine order & orientation of the Ψs
o Studying chromosome homeology, genome and karyotype evolution
within species of the same and other genera of Lemnoideae by means of
comparative chromosome painting
o Establishing an optimized system/pipeline enabling a quick detection of
chromosomal rearrangements in duckweed species/clones
Acknowledgement IPK, Gatersleben, Germany
o Ingo Schubert
o Joerg Fuchs
o Martina Kuehne
o Joachim Bruder
o Rocío Rubio
University of Jena, Germany
o Klaus Appenroth
Waksman Institute of Microbiology,
Rutgers University, USA
o Wenqin Wang
o Joachim Messing
Spirodela polyrrhiza (L.) Schleid.
[as Lemna polyrrhiza L.]
Sowerby, J.E., English Botany, or
Coloured Figures of British Plants,
3th ed., vol. 9: t. 1397 (1869)
