Gen om e d iver si ty

Goals
Genomic and transcriptomic analyses to identify candidate 
apomixis factors in wild monocot and dicot species.

Abteilung Cytogenetik und Genomanalyse/
Department of Cytogenetics and Genome Analysis
76
(2) the hexaploids have been repeatedly generated from tetra-
ploids during the course of evolution.  interestingly, tetraploids 
are highly apomictic, while secondarily-derived hexaploids are 
highly sexual, providing evidence for dosage of a factor affect-
ing the penetrance of apomixis.  
The Ranunculus auricomus complex: using illumina technol-
ogy, we have sequenced the complete transcriptomes of  
2 apomictic and 3 sexual Ranunculus genotypes, and are 
using this data to design a 3 x 1.4 million feature custom 
(NimbleGen) expression microarray.  Live ovules from 4 de-
velopmental stages and 5 accessions each of sexual and apo-
mictic Ranunculus have been microdissected and a compara-
tive transcriptomal profiling experiment has been completed. 
Over 100 000 high quality SNPs have been mined from the 
sequence data, and dn/dS ratios between sexual and apo- 
micts imply that divergent selection has acted on genes associ-
ated with the switch from sex to apomixis. 
Poa pratensis: in our goals to identify apomixis factors in mono-
cots, we have initiated a project in the kentucky bluegrass (Poa 
pratensis). using illumina technology, we have sequenced the 
complete transcriptomes each of an apomictic and sexual 
Poa pratensis genotype, and have used this data to design a 3 
x 720K feature custom (NimbleGen) expression microarray. 
Live ovules from a single developmental stage from 10 geno-
types have been microdissected and a comparative transcrip-
tomal profiling experiment has been completed. these data are 
in the analysis step.
We have designed a 3 x 750 000 feature custom (NimbleGen) 
comparative genome hybridization (CGH) microarray based 
upon the sequenced cdna libraries, and have completed a copy 
number variation (cnV) experiment on 10 different genotypes 
each of sexual and apomictic Boechera genotypes. importantly, 
these data demonstrate that the apomixis phenotype has been 
expressed repeatedly from different sexual backgrounds, a re-
sult which is consistent with the hypothesized spread of aPoL-
Lo and uPGRade-2 via interspecific hybridization.
in a separate project, we have sequenced 6 genomes each of 
diploid sexual and diploid apomictic Boechera which occur 
sympatrically in colorado, with the goal of measuring deleteri-
ous mutation accumulation (i.e. Muller’s ratchet) which is ex-
pected to occur as a result of asexuality. these data have been 
analysed with respect to phylogenetically distant taxa in order 
to identify conserved nucleotides (i.e. those with no change 
across all taxa and sexual Boechera). these conserved nucleo-
tide positions were subsequently analysed in all apo mictic ge-
nomes, and demonstrate strong evidence for mutation accu-
mulation at many thousand sites (Fig. 25).  
Hypericum perforatum: We have chosen 650 accessions, rep-
resenting different ploidies and worldwide geographic origins, 
for which data from 30 microsatellite markers have been ana-
lysed. a flow cytometric analysis of 96 seeds per each acces-
sion demonstrates genotype-specific quantitative variation 
for sexual and apomictic seed production. these data demon-
strate (1) that H. perforatum colonized north america from 3 
distinct european genetic lineages over the last 160 years, and 
Fig. 25
consistently elevated ratio of 
conserved noncoding to 4-fold 
neutral heterozygous sites 
in apomictic Boechera  from 
different populations (x-axis) 
 
(J. Lovell, t. Sharbel).

77
Publications
Peer Reviewed Papers
2012
c
ockram
, J., t. t
hiel
, b. S
teuernagel
, n. S
tein
, S. t
audien
, P.c. b
ailey
 & 
d.M. o’S
ullivan
: Genome dynamics explain the evolution of 
flowering time cct domain gene families in the Poaceae. 
PLoS one 7 (2012) e45307.
R
óis
, a.S., G. t
eixeira
, t.F. S
harbel
, J. F
uchs
, S. M
artins
, d. e
spírito
-S
an
-
to
 & a.d. c
aperta
: Male fertility vs sterility, cytotype and dna 
quantitative variation in seed production in diploid and 
tetraploid sea lavenders (Limonium sp., Plumbaginaceae) 
reveal diversity in reproduction modes. Sex. Plant Reprod. 
25 (2012) 305-318.
V
oigt
-z
ielinski
, M.-L., M. P
iwczynski
 & t.F. S
harbel
: differential effects 
of polyploidy and diploidy on fitness of apomictic Boechera. 
Sex. Plant Reprod. 25 (2012) 97-109.
2013
a
liyu
,  o.M., M. S
eifert
, J.M. c
orral
, J. F
uchs
 & t.F. S
harbel
: copy 
number variation in transcriptionally active regions of sex-
ual and apomictic Boechera demonstrates independently-
derived apomictic lineages. Plant cell 25 (2013) 3808-3823.
a
miteye
,  S., J.M. c
orral
, h. V
ogel
, M. k
uhlmann
, M.F. M
ette
 & t.F. 
S
harbel
: novel microRnas and microsatellite-like small 
Rnas in sexual and apomictic Boechera species. MicroRna 
2 (2013) 46-63.
c
orral
, J.M., h. V
ogel
, o.M. a
liyu
, G. h
ensel
, t. t
hiel
, J. k
umlehn
 & 
t.F. S
harbel
: a conserved apomixis-specific polymorphism 
is correlated with exclusive exonuclease expression in pre-
meiotic ovules of apomictic Boechera species. Plant Physiol. 
163 (2013) 1660-1672.
de
 S
torme
, n., L. z
amariola
, M. M
au
, t.F. S
harbel
 & d. G
eelen
: Volume-
based pollen size analysis: an advanced method to assess 
somatic and gametophytic ploidy in flowering plants. Plant 
Reprod. 26 (2013) 65-81.
d
obeš
,  c., a. M
ilosevic
, d. P
rohaska
, S. S
cheffknecht
, t.F. S
harbel
 & 
k. h
ülber
: Reproductive differentiation into sexual and apo-
mictic polyploid cytotypes in Potentilla puberula (Potentil-
leae, Rosaceae). ann. bot. 112 (2013) 1159-1168.
G
alla
,  G., M. V
olpato
, t.F. S
harbel
 & G. b
arcaccia
: computational 
identification of conserved microRnas and their putative 
targets in the Hypericum perforatum L. flower transcrip-
tome. Plant Reprod. 26 (2013) 209-229.
h
ofrichter
, J., M. k
rohn
, t. S
chumacher
, c. L
ange
, b. F
eistel
, b. W
al
-
broel
, h.-J. h
einze
, S. c
rockett
, t.F. S
harbel
 & J. P
ahnke
: Reduced 
alzheimer’s disease pathology by St. John’s wort treatment 
is independent of hyperforin and facilitated by abcc1 and 
microglia activation in mice. curr. alzheimer Res. 10 (2013) 
1057-1069.
k
och
, M.a., c. S
cheriau
, a. b
etzin
, n. h
ohmann
 & t.F. S
harbel
: evo-
lution of cryptic gene pools in Hypericum perforatum: the 
influence of reproductive system and gene flow. ann. bot. 
111 (2013) 1083-1094.
L
ovell
,  J.t., o.M. a
liyu
, M. M
au
, M.e. S
chranz
, M. k
och
, c. k
iefer
, 
b.h. S
ong
, t. M
itchell
-o
lds
 & t.F. S
harbel
: on the origin and 
evolution of apomixis in Boechera. Plant Reprod. 26 (2013) 
309-315.
M
au
, M., J.M. c
orral
, h. V
ogel
, M. M
elzer
, J. F
uchs
, M. k
uhlmann
, n. 
de
  S
torme
, d. G
eelen
 & t.F. S
harbel
: the conserved chimeric 
transcript UPGRADE-2 is associated with unreduced pollen 
formation and is exclusively found in apomictic Boechera. 
Plant Physiol. 163 (2013) 1640-1659
.
P
ellino
,  M., d. h
ojsgaard
, t. S
chmutzer
, u. S
cholz
, e. h
örandl
,  
h. V
ogel
 & t.F. S
harbel
: asexual genome evolution in the 
apo mictic  Ranunculus auricomus complex: examining the 
effects of hybridization and mutation accumulation. Mol. 
ecol. 22 (2013) 5908-5921.
P
iwczyński
, M., a. P
onikierska
, R. P
uchalka
 & J.M. c
orral
: expression 
of anatomical leaf traits in homoploid hybrids between de-
ciduous and evergreen species of Vaccinium. Plant biol. 15 
(2013) 522-530.
PhD and Diploma Theses
2012
a
miteye
, S.: MicroRnas profiling and analysis in floral tissues of 
sexual and apomictic Boechera species. (Phd thesis) Ru-
precht-karls-universität, heidelberg und international Max 
Planck Research School (iMPRS), Jena (2012) 153 pp.
2013
L
ovell
, J.: What are the factors that constrain or promote adapta-
tion in Arabidopsis thaliana and its close relative, Boechera? 
(Phd thesis) colorado State university – Fort collins/uSa 
(2013) 138 pp.
M
au
, M.: identification, molecular analysis and phylogeograph-
ic distribution of the chimeric UPGRADE-2 gene, a candi-
date for the initiation of unreduced pollen formation in 
apomictic Boechera (brassicaceae). (Phd thesis) Ruprecht-
karls-universität, heidelberg und international Max Planck 
Research School (iMPRS), Jena (2013) 168 pp.
Patents
2012
s
harbel
, t. & J. c
orral
-g
arcia
: Means and methods to induce apo-
mixis in plants. Wo 2012/163818, offenlegung: 6.12.2012, 
iPk-nr. 2010/02.

Abteilung Cytogenetik und Genomanalyse/
Department of Cytogenetics and Genome Analysis
78
utilising a transgene-based reporter system showing parti-
cularly high sensitivity to RdtGS, a forward genetic screen for 
eMS-induced no rna-directed transcriptional silencing (nrd) mu-
tants was performed in Arabidopsis thaliana in order to identify 
more components of the RdtGS/RddM pathway. as we repor-
ted in epigenetics, three of the obtained nrd mutant lines con-
tained novel idn2/rdm12 and nrpd2a/nrpe2a loss-of-function 
alleles.  IDN2/RDM12 encodes a Xh/XS domain protein that is 
able to bind double-stranded Rna with 5’-overhangs, while 
NRPD2a/NRPE2a encodes the common second-largest subunit 
of dna-dependent Rna polymerases Pol iV and V involved in 
silencing. both, idn2/rdm12 and nrpd2a/nrpe2a, were found to 
release target transgene expression and reduce chh context 
methylation at transgenic as well as endogenous RddM target 
regions to a similar extent. nevertheless, accumulation of in-
verted repeat-derived siRnas was not affected, allowing us to 
establish a refined model of RddM that positions function of 
IDN2 downstream of siRNA formation and points to an im-
portant role for its Xh domain (F. Finke, M.F. Mette). 
Further, in a cooperative project to harness minichromosomes 
as shuttles for recombinant genes in plants, de novo formation 
of centromeres via sequence-specific tethering of cenh3 was 
approached (c.h. teo, M.F. Mette, in collaboration with research 
groups karyotype evolution and chromosome Structure and 
Function). 
Publications
Peer Reviewed Papers
2012
F
inke
, a., M. k
uhlmann
 & M.F. M
ette
: idn2 has a role downstream 
of siRna formation in Rna-directed dna methylation. epi-
genetics 7 (2012) 950-960.
F
inke
, a., F.M. M
ette
 & M. k
uhlmann
: Genetic analysis of Rna-me-
diated gene silencing in Arabidopsis thaliana. J. Verbr. Le-
bensm. 7 (2012) 27-33.
k
apusi
, e., L. M
a
, c.h. t
eo
, G. h
ensel
, a. h
immelbach
, i. S
chubert
, F.M. 
M
ette
, J. k
umlehn
 & a. h
ouben
: telomere-mediated truncation 
of barley chromosomes. chromosoma 121 (2012) 181-190.
k
uhlmann
,  M. & M.F. M
ette
: developmentally non-redundant 
Set domain proteins SuVh2 and SuVh9 are required for 
transcriptional gene silencing in Arabidopsis thaliana. Plant 
Mol. biol. 79 (2012) 623-633.
Scientists
IPK financed
Finke, andreas (0,50, till 31.12.2012)
teo, chee how, dr. (till 30.04.2012)
Grant Positions
kuhlmann, Markus, dr. (dFG/SFb 648, till 31.12.2012)
Goals
analysis and utilisation of epigenetic control mechanisms act-
ing at the chromatin level to warrant regulation and structural 
maintenance of plant genomes.
Research Report
the research group epigenetics was closed down by the end of 
december 2012. 
Short interfering (si) Rnas can act as nucleotide sequence-
specific regulators of gene expression at the transcriptional as 
well as the post-transcriptional level. in plants, siRnas targeting 
promoter regions can induce RNA-directed transcriptional 
gene silencing (RdTGS) correlated with RNA-directed DNA 
methylation (RdDM). 
by a reverse genetic approach in Arabidopsis thaliana, we could 
demonstrate in a Plant Molecular biology article that two close-
ly related members of the suppressor of variegation 3-9-homo-
logous (SUVH) group of putative histone methyltransferases, 
SUVH2 and SUVH9, act partially non-redundant in RdDM. 
dna methylation analysis at the endogenous RddM-target 
AtSN1 (a SINE-like retroelement) in suvh2 and suvh9 single mu-
tants revealed that SuVh2 is required for full AtSN1  methyla tion 
during early seed development, while SuVh9 has its impact on 
RddM later during vegetative development of the plant. how-
ever, in a suvh2 suvh9 double mutant stronger reduction of 
AtSN1 methylation was found than in any of the single mutants. 
also histone 3-lysine 9-dimethylation (h3k9me2) associated 
with AtSN1 was reduced only in the simultaneous absence of 
functional SuVh2 and SuVh9. thus, SuVh2 and SuVh9 seem to 
have overlapping functions in RddM in spite of some develop-
mental specialisation. as transcripts of the plant-specific dna-
dependent Rna Pol V were reduced in suvh2 suvh9 double 
mutants, SuVh2 and SuVh9 might have roles in Pol V complex 
recruitment (M. kuhlmann, M.F. Mette). 
Research Group: Epigenetics 
(till 31 december 2012)
head:  dr. Michael Florian Mette

79
2013
a
miteye
,  S., J.M. c
orral
, h. V
ogel
, M. k
uhlmann
, M.F. M
ette
 & t.F. 
S
harbel
: novel microRnas and microsatellite-like small 
Rnas in sexual and apomictic Boechera species. MicroRna 
2 (2013) 46-63.
h
ouben
, a., M.F. M
ette
, c.h. t
eo
, i. L
ermontova
 & i. S
chubert
: engi-
neered plant minichromosomes. int. J. dev. biol. 57 (2013) 
651-657.
L
ermontova
,  i., M. k
uhlmann
, S. F
riedel
, t. R
utten
, S. h
eckmann
, M. 
S
andmann
, d. d
emidov
, V. S
chubert
 & i. S
chubert
: Arabidopsis ki-
netochoRe nuLL
2
 is an upstream component for cenh3 
deposition at centromeres. Plant cell 25 (2013) 3389-3404.
t
eo
, c.h., i. L
ermontova
, a. h
ouben
, M.F. M
ette
 & i. S
chubert
: de novo 
generation of plant centromeres at tandem repeats. chro-
mosoma 122 (2013) 233-241.
PhD and Diploma Theses
2012
b
ruchmüller
,  a.: untersuchungen zur post-transkriptionellen 
Gen-inaktivierung in (mono kotylen und dikotylen) Pflan-
zen und deren beeinflussung zur erhöhung der transgen-
expression. (Phd thesis) Martin-Luther-universität halle-
Wittenberg, institut für agrar- und ernährungswissenschaf-
ten der naturwissenschaftlichen Fakultät iii, halle/S. (2012) 
120 pp.
2013
F
inke
, a.: Genetic and functional characterization of Rna-direc-
ted transcriptional gene silencing in Arabidopsis thaliana. 
(Phd thesis) Martin-Luther-universität halle-Wittenberg, 
naturwissenschaftliche Fakultät i, biowissenschaften, 
halle/S. (2013) 131 pp.

Abteilung Cytogenetik und Genomanalyse/
Department of Cytogenetics and Genome Analysis
80
actions with the barley powdery mildew B. graminis f.sp. hordei, 
possibly interfering with the enforced maintenance of plastid 
function in powdery mildew-colonized cells. Rnr6 encoding a 
cellulose synthase-like protein of the d clade (HvCSLD2) was 
found to be important for both quantitative host- as well as non-
host resistance. however, progress was slow because the protein 
turned out to be extremely difficult to express in yeast for enzy-
matic activity studies, and because cell-wall alterations in trans-
genic CSLD2 Rnai lines appear to be subtle, at least as long as 
local changes at penetration sites are not looked at. Finally, Rnr8 
encoding a leucine-rich repeat-containing receptor kinase 
(RLK) plus three other RLk’s with converging evidence for a role 
in nonhost resistance were selected for site-directed mutagene-
sis by taLen technology in barley and for transgenomic comple-
mentation in transgenic wheat, in collaboration with J. kumlehn/
research group Plant Reproductive biology and L. boyd/niab. 
From the tiGS screening of factors affecting race-nonspecific 
host resistance we focus on three genes and alleles encoding the 
zinc-finger protein HvLsd1a, the germin-like protein HvGER4 
and a novel proline-rich protein with structural similarity to the 
rice resistance gene pi21. For all three, transgenic Rnai lines have 
been analysed and confirmed a role in barley-pathogen interac-
tions (a. himmelbach and d. douchkov).
Biotechnology for pathogen-stress resistance in barley and wheat:
From the shortlist of approximately 40 barley candidates with 
converging evidence for an important role in race-nonspecific 
resistance we selected 22 for (i) gene marker-assisted single 
and multiple backcrossing, (ii) triple overexpression of putative 
resistance-mediating genes, and (iii) triple Rnai of putative sus-
ceptibility factorys (all driven by pathogen-inducible hvGeR4 
promoters) in transgenic barley with emphasis on cell-death 
regulators and cell-wall modifying proteins. novel binary vec-
tors were produced and tested, plant material was generated 
in close collaboration with partners from the baRLeY-fortress 
consortium, and resistance phenotyping of transgenic- and 
backcross lines is currently in progress in the greenhouse and 
will be performed in field trials in 2014, respectively (d. nowara 
and d. douchkov). host-induced gene silencing (hiGS) in fun-
gal pathogens is a promising approach to durable resistance. 
the largest effort was invested into improving resistance of 
barley and wheat against Fusarium culmorum, one of the Fusar-
ium head blight (Fhb) fungi. For this aim, transient and stable 
silencing by the barley-stripe mosaic virus (bSMV) and in trans-
genic plants, respectively, was tested. among other hiGS tar-
gets, fungal glucan synthase 1 (FcGS1) appeared promising and 
resulted in enhanced resistance in the greenhouse as well as 
under field-like conditions, besides phenocopying cell-wall ab-
normalities described for stable fungal GS1 Rnai lines (W. chen 
and d. nowara).
PROGRAMME: FUNCTIONAL GENOME ANALySIS
Research Group: Pathogen Stress Genomics
head:  dr. Patrick Schweizer
Scientists
IPK financed
Rajaraman, Jeyaraman (0,50, since 03.08.2013)
Grant Positions
chen, Wanxin, dr. (bMbF, till 31.07.2013)
douchkov, dimitar, dr. (bMbF, till 30.09.2012; bMbF/dPPn, 
since 01.10.2012)
hasan, Mainul (0,50 bMbF, till 31.03.2012)
himmelbach, axel, dr. (0,75 bMbF, till 30.04.2010)
nowara, daniela, dr. (Wirtschaft-923920, till 14.10.2012 ; bMbF, 
since 15.10.2012)
Razzak, hassan (0,50 bMbF, since 01.08.2012)
Visiting Scientists/Scholars
chen, Wanxin, dr. (self-financed, since 01.07.2013)
hu, Wenjin (daad, 07.09.-30.11.2013)
ihlow, alexander, dr. (th ilmenau, since 01.01.2012)
zhang, tingting (daad, 07.09.-30.11.2013)
Goals
Genes, pathways and biotechnology for pathogen-stress resist-
ance in cereals, and integration with abiotic-stress responses.
Research Report
Genes and alleles for pathogen-stress resistance in barley:
in recent years we have performed a number of functional 
genomics screens in barley attacked by adapted or non-adapt-
ed (nonhost) powdery mildew fungi. this led to the discovery 
of a shortlist of approximately 40 gene candidates with con-
verging evidence for an important role in PaMP-triggered im-
munity during host or nonhost interactions (see progress re-
port 2010/2011). out of these, we currently focus on nine genes 
of outstanding interest, as briefly outlined below.
three prioritized candidates were derived from a tiGS (transient 
induced Gene Silencing) screening in barley epidermal cells at-
tacked by the wheat powdery mildew fungus Blumeria graminis 
f.sp.  tritici.  Rnr5 (Required for Nonhost Resistance 5) encoding 
HvARM1, a partial duplicate of the U-box ARM-repeat E3 li-
gase hvPub15 turned out to be more relevant for the host- than 
for the nonhost interaction. Recent data from transient expres-
sion or tiGS of HvARM1, transgenic HvARM1 Rnai lines as well 
as in vitro and in vivo protein-protein interaction studies suggest 
that the partial gene duplicate plays a role as decoy of the sus-
ceptibility-related e3-ligase HvPUB15 in susceptible host inter-

81
www.pflanzenforschung.de/de/journal/journalbeitrage/
neue-perspektiven-fuer-den-pflanzenschutz-mit-higs-in-
te-10040 (2013).
R
edaktion
 P
flanzenforschung
.
de
: Projektporträt “dsRnaguard”. kul-
turpflanzen schalten die Gene ihrer Feinde ab. http://www.
pflanzenforschung.de/de/journal/journalbeitrage/ projekt-
portraet-dsrnaguard-kulturpflanzen-schalten-die-10041 
(2013).