Trude Schwarzacher: #ECA2015 European Cytogenetics Conference plenary talk:150 years since Mendel's laws of heredity
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Mendel and his time in the light of cytogenetics
Trude SchwarzacherUniversity of LeicesterDepartment of GeneticsTS32@le.ac.uk
Gregor Mendel (1822-1884)
www.molcyt.com
UserID/PW ‘visitor’
Hugo Iltis‘Few publications have so enduringly and
variously influenced science as had the short
monograph [Versuche über Pflanzenhybriden] by
the Augustinian monk of Brünn [now Brno], Pater
Gregor Mendel. Forgotten for decades, within a
few years after its rediscovery it gave a
mighty impetus to the doctrine of heredity;
and as Mendelism, his teaching had now become
the central theme of biological research as well as
the foundation of manifold practical application’
Mendel’s life
(The Life of Mendel,1966)
Mendel’s life Mendel’s experiments Why was he forgotten? What was known about chromosomes
at the time of Mendel? Mendel’s rediscovery at 1900 How has chromosome biology
developed since Some examples from our own
research related to Mendel and plant hybridization
Overview
20 July 1822: born as Johann Mendel, Heinzendorf bei Odrau, Austrian Empire (now Hynčice, Czech Republic)1840 – 1843: practical and theoretical philosophy and physics at the University of Olomouc
Mendel’s life
1843: joined as Pater Gregor the Augustinian Monastery, Brünn (now Brno) 1847: ordained priest1851-1853: Natural history at the University of Vienna under Franz Ungar (professor of plant physiology) and Christian Doppler (professor of physics) 1853 onwards: supply teacher at Brno; he failed the exam to become a certified teacher twice1857-1864: Experiments with peasSpring 1865: presented the results and generalizations at two meetings of the Natural History Society of Brünn
Mendel’s life
1866: The papers were printed in the Proceedings of the Society distributed in Europe and America1866: Mendel consults Karl Wilhelm Nägeli of Munich, leading botanist of the time. Nägeli does not understand the significance of Mendel’s results and laws of heredity1868: Becomes abbot; and has decreasing time for scientific activities1869: Results on Hieraceum6 January 1884: died at the age of 61
Mendel’s life
Mendel Memorial in Brno
Mendel’s life
Pat Heslop-Harrison in 2001
Jack Heslop-Harrison in 1933
Mendel’s life Mendel’s experiments Why was he forgotten? What was known about chromosomes
at the time of Mendel Mendel’s rediscovery How has chromosome biology
developed since Some examples from our own
research
Overview
But Mendel does not mean hybrids between two species, he means between two different types or variants
“…the regularity with which the same hybrid forms resulted,every time fertilization between the same species occurred, gave the incentive to further controlled experiments.”
Experiments with peasCrossing (making hybrids with) varieties with clear and different characters or traits
Drawing from many websites including http://guestblog.scientopia.org/2012/08/03/mud-sticks-especially-if-you-are-gregor-mendel/
Experiments with peas
Law of segregation
Experiments with peas
Law of independent
assortment
Experiments with peas Defined the terms recessive and dominantSpoke of invisible ‘factors’ -now called genes – that were responsible for the visible traits
Genetic location of Mendel's seven characters on pea linkage groups. Yellow versus green cotyledons II/ii on linkage group (I); seed coat (and flower) colour AA/aa on linkage group (II); tall versus dwarf plants (LeLe/lele) on linkage group (III); difference in the form of the ripe pods (PP/pp or VV/vv) on linkage groups (III) and (VI), respectively; difference in the position of the flower (FasFas/fasfas or FaFa/fafa) on linkage groups (III) or(IV), respectively; round versus wrinkled (RR/rr) on linkage group (V); and colour of unripe pod (GpGp/gpgp) on linkage group (V).
The types of lesion in Mendel's mutants are various: transposon insertion (r), missense mutation (le-1), splice variant (a) and amino acid insertion (i).
Ellis et al. 2011: Mendel, 150 years on. Trends in Plant Science 11:590-596.
2010, doi:10.1371/journal.pone.0013230
Conclusions/Significance: Identification of the pea genes A that is the factor determining
anthocyanin pigmentation in pea The A gene encodes a bHLH transcription factor. The white flowered mutant allele most likely used by Mendel is
a simple G to A transition in a splice donor site that leads to a mis-spliced mRNA with a premature stop codon
The wrinkled-seed mutant (rr) of pea (Pisum sativum L.) arose through mutation of the gene encoding starch-branching enzyme isoform I (SBE1) by insertion of a transposon-like element into the coding sequence. Starch amount and amylopectin are reduced and as a consequence sucrose level is higher that causes increased uptake of water. When the seed tries the wrinkled phenotype results.
Mendel’s life Mendel’s experiments Why was he forgotten? What was known about chromosomes
at the time of Mendel Mendel’s rediscovery How has chromosome biology
developed since Some examples from our own
research
Overview
Mendel’s experiments
Mendel’s 1866 paper was cited only three times over the next thirty-five years.
Was Mendel ahead of his time? Did he have bad luck?
Boring title and Mendel did not sell his theory well.
Mislead by Nägeli to work on Hieraceum that did not prove his theory
His paper was seen as essentially about hybridization rather than inheritance
His paper was seen as essentially about hybridization rather than inheritance.
Blended inheritance was the accepted theory of inheritance where traits from each parent are averaged together. not so different from what we now know to be
the case for multiple genes and quantitative trait loci (QTL).
Mendelian discontinuous inheritance applies to single genes only.
Inheritance
Mendel’s life Mendel’s experiments Why was he forgotten? What was known about
chromosomes at the time of Mendel
Mendel’s rediscovery How has chromosome biology
developed since Some examples from our own
research
Overview
micro.magnet.fsu.edu/primer/museum/hooke.html
First microscope observed cells
Compound microscope
The lens closest to the object, called the Objective, is used to enlarge and invert the object into a 'real' image.
The lens closest to the eye called the Eyepiece or Ocular acts essentially as a simple magnifier, used to view the image formed by the objective. The simple magnifier is a converging lens placed in front of the eye that increases the size of the image formed by the retina.
www.math.ubc.ca/.../lewis/project.html
The compound microscope, in its simplest form is a system of two converging lenses used to look at very small objects at short distances.
E. Leitz, Wetzlar, Germany, 1894
Modern microscope
www.math.ubc.ca/.../lewis/project.html
E. Leitz, Wetzlar, Germany, 1894
Axioimager
Recording chromosome images
Drawings Photography (1950-1980s)
Black and white Colour
Digital images (1990s, now) CCD cameras
www.usyd.edu.au/.../cmicrodesign.shtml
http://www-unix.oit.umass.edu/~coreya/yashica/micadpt.jpg
First photograph: 1826,Eastmann (1884): film as known today. Microphotography 1920/30.
Chromosomes
Early 19th centuryCells and nuclei simply pinched in half to divide
Anton Schneider (1873)
First scientist to describe clearly the process of mitosis and the involvement of the ‘chromatic nuclear figure’
Eduard Strassburger (1875) Gives clear and detailed descriptions of cell division in plants
Walther Flemming (1879-1882)Describes ‘Mitosis’ in animal cellsDiscovers lampbrush chromosomes
Balbiani (1880)Polytene chromosomes
Chromosome
Flemming 1882
Continuity of chromosomes throughout cell division
Flemming 1882
Orientation of chromosomes within interphase Rabl (1885) Rabl orientation
Salamandra maculata
1B/1R wheatWheat containing chromosome 1RS
Schwarzacher et al. 1992
Metaphase I
Telomere
Centromeres
Interphase
Rye
Schwarzacher 2000
Gregor Mendel (1865)Formulated his laws of heredity without
the knowledge of chromosomes
Wilhelm Waldeyer (1888)Introduces the term ‘chromosome’
Weismann (1887)Puts forward ‘chromosome theory of
inheritance’
1900: When Mendel was rediscovered, it became clear that the behaviour of chromosomes at cell division (mitosis and particular meiosis) was exactly what was needed to explain the distribution of hereditary factors
Waldeyer
Who and how was he rediscoveredMendel’s laws were rediscovered independently within two months of each other in Spring of 1900 by Hugo de Vries and Carl Correns, and to some extend the Austrian Erich von Tschermak. Following their publications, Mendel’s results were replicated and genetic linkage formally described.
Rediscovery
Mendel’s life Mendel’s experiments Why was he forgotten? What was known about chromosomes
at the time of Mendel Mendel’s rediscovery How has chromosome biology
developed since Some examples from our own
research
Overview
Bateson (1916)Described the concept of the gene
Feulgen and Rossenbock (1924)
Demonstrated the presence of DNA in chromosomes by histochemical staining
Watson and Crick (1953) Structure of DNA
Early studies on chromosomes were in insects and plants
Morgan and his students (Drosophila; linkage groups)Barbara McClintock (maize, transposable elements)
Number of chromosomes in human was not established until
1956 (J. Tijo and A. Levan)
Chromosomes, genes and DNA
Fluorescent in situ hybridization (FISH)
FISH 1985 onwards
Beta vulgaris
Propidium idodeFITC
Images from molcyt.com (upper row) and chrombios.com (lower row)
Wheat, Hieraceum and Petunia
Polyploid and diploid hybrids
Oil seed rape, Brassica napus
Petunia hybrida
P. axillaris
Hieraceum
Wheat trials
Triticalewheat x rye
hybrid
Schwarzacher et al 1989, 1992Total genomic DNA labels chromosomes according to their genome origin
2n=6x=42AABBRR
Annals of Botany 64, 315-324 and Theoretical and Applied Genetics 84, 778-786..
Rye and the genus Thinopyrum, including wild goat grasses and wheat grasses, has proven an excellent source for disease and biotic stress resitance
Schwarzacher 2000
Six populations of wheat lines that include an alien chromosome arm from Thinopyrum intermedium carrying WSMV resistance (Wsm-1 gene)
Characterization of new sources of Wheat streak mosaic virus resistance
WSMV resistant and susceptible lines in field trials
Bob Graybosch, USDA-ARS, University of Nebraska, USAWheat ‘Mace’: Journal of Plant Registrations 3(1): 51-56.doi: 10.3198/jpr2008.06.0345crc
4D T4DL*4Ai#2S
DAPI Afa Thin all(blue) (green) (red)
Some lines also carry a Thin or rye fragment on
chromosome 1B
Th. intermedium DNA
pSc119.2/CS13
Rye DNAdpTa1/Afa
The whole 1RS arm correlates with WSMV resistance in the absence of 4D and when together with 4D enhances resistance
Ali, Graybosch, Hein, Heslop-Harrison, and Schwarzacher 2015
Hieraceum• Genus Hieraceum• Hawkweed (German Habichtskraut)• Family Asteraceae (Compositae)• Closely related to Taraxacum (Dandelion)• Probably 1,000+ species• Classification notoriously difficult with a lot of minor
geographical variation
Most reproduce exclusively asexually by means of seeds that are genetically identical to their mother plant (apomixis or agamospermy)
Rubar Salih, Richard Gornall and Pat Heslop-Harrison
HieraceumTaxon Section Chr
numberPloidy
Identifier code
Source
H.amaurostictum Walter Scott & R.C.Palmer
Alpestria 2n=36 4x Hama01 Semblister
H.attenuatifolium P.D.Sell & C.West
Alpestria 2n=36 4x Hatt02 Laxo Burn
H.australis (Beeby)Pugsley Alpestria 2n=36 4x Haus03 Burrafirth area, UnstH.breve Beeby Alpestria 2n=36 4x Hbre04 Ronas VoeH.difficile P.D.Sell & C.West Alpestria 2n=36 4x Hdif05 OkraquoyH.dilectum P.D.Sell & C.West Alpestria 2n=36 4x Hdil06 LaxoH.gratum P.D.Sell & C.West Alpestria 2n=36 4x Hgra08 Burra Firth, UnstH.hethlandiae (F.Hanb.) Pugsley
Alpestria 2n=36 4x Hhet09 Mavis Grind
H.northroense Pugsley Alpestria 2n=27 3x Hnor11 Burravoe, North RoeH.pugsleyi P.D.Sell & C.West Alpestria 2n=36 4x Hpug12 Whale Firth, YellH.spenceanum Qalter Scott & R.C.Plamer
Alpestria 2n=36 4x Hspe14 Sandness
H.subtruncatum Beeby Alpestria 2n=36 4x Hsubt16 Scarvister, West Mainland
H.vinicaule P.D.Sell & C.West Alpestria 2n=27 3x Hvin17 Whale FirthH.zetlandicum Beeby Alpestria 2n=36 4x Hzet18 Isbister, North RoeH.scottii P.D.Sell Oreadea 2n=36 4x Hsco13 Near Windy ScordH.subscoticum P.D.Sell Oreadea 2n=27 3x Hsubs15 Ronas VoeH.gothicoides Pugsley Tridentata 2n=37 4x Hgot07 LunningH.lissolepium Roffey Tridentata 2n=36 4x Hlis10 Eric’s Ham, YellH.umbellatum _ 2n=18 2x Humbi19 Sw/71.50
Samples supplied by Richard Gornall, Botanic Garden, University Leicester
HieraceumBarcoding
Chloroplast Matk geneITS of the 45S rDNA
Rubar Salih, Richard Gornall and Pat Heslop-Harrison
H. vinicaule H17 (2n=3x= 27)
Genomic in situ hybridization with Hieracium umbellatum DNA
H. northroense H11 (2n=3x= 27)
Rubar Salih and Pat Heslop-Harrison 2014
FISH with rDNA probes3x species have 3 or 6 sites4x species have 4 or 8 sites
H. vinicaule H17 (2n=3x= 27) H. amaurostictum H1 (2n=4x= 36)
45S rDNA 45S rDNA 5S rDNA
Rubar Salih and Pat Heslop-Harrison 2014
K. Richert Poeggeler and Schwarzacher
Diploid hybrid2n=14
Petunia is a model for DNA transposon work
P. hybrida
Petunia inflata X P. axillaris2n=14 x 2n=14
1. Transposon insertion, blocks the colour production in the floral pigment pathway
2. Spontaneous excision of elements restores colour and causes variegation
Extremely active endogenous dTph1 transposon system
Gerats, A.G., Huits, H., Vrijlandt, E., Marana, C., Souer, E., and Beld, M. (1990). Molecular characterization of a nonautonomous transposable element (dTph1) of petunia. Plant Cell 2, 1121-1128. http://solgenomics.net/community/feature/200601.pl
Petunia Genome Consortium Petunia Leader Cris Kuhlemeier with Quattrocchio, Sims, Mueller, Schranz, Bombarely,Richert-Pöggeler, Schwarzacher, Heslop-Harrison et al.
P. inflata P. hybrida P.axillaris
http://flower.ens-lyon.fr/PetuniaPlatform/Petunia_as_a_model.html
Solanaceae phylogenyTomato Potato Tobacco
Eric Schranz and Trude Schwarzacher 2015 adapted from
Sarkinen, T., Bohs, L., Olmstead, R.G., and Knapp, S. (2013). A phylogenetic framework for evolutionary study of the nightshades (Solanaceae): a dated 1000-tip tree. BMC evolutionary biology 13, 214.
Repetitive DNA component in Petunia
Petunia Consortium 2015
Organelle sequencesfrom chloroplasts or
mitochondria
Sequences from viruses, Agrobacterium or other
vectors
Transgenes introduced with molecular biology
methods
Genes, regulatory and non-coding single copy sequences
Dispersed repeats:Transposable Elements
Repetitive DNA sequences
Plant Nuclear Genome
Tandem repeats
DNA transposons copied and
moved via DNA
Retrotransposons amplifying via an RNA intermediate
Centromeric repeats
Structural components of chromosomes
Telomeric repeats
Simple sequence repeats or
microsatellites
Repeated genes
Subtelomeric repeats
45S and 5S rRNA genes
Blocks of tandem repeats at discrete chromosomal loci
DNA sequence components of the plant nuclear genomeHeslop-Harrison & Schmidt 2012. Encyclopedia of Life Sciences
Other genes
Horizontal DNA transfer
Petunia Vein Clearing Virus (PVCV, 7206bp)
Richert-Poeggeler and Shepherd 1997Virology 236, 137-146
Para-retrovirusDoes not need genomic integration for replication
PVCV
ePVCV
metaviridae-like sequencesPVCV
QT
R
integrase +2
pol +3
gag-pol +2
l clone3 (8 kb)
6170-7198
+2
metaviridae-like sequences
PVCV (nt 665-6153) +3gag-pol -1
+1
l clone 4 (11.4 kb)
K. Richert Poeggeler, J. Baily and SchwarzacherMetaviridae
PVCV: Petunia vein clearing virus ePVCV are present and are clustered with Ty3-gypsy-like sequences
Richert-Pöggeler, K.R., Noreen, F., Schwarzacher, T., Harper, G. and Hohn, T. (2003) Induction of infectious Petunia vein clearing (pararetro) virus from endogenous provirus in petunia. EMBO Journal 22: 4836-4845
PVCV
K. Richert Poeggeler and Schwarzacher
2000YA
methylated DNA
unmethylated DNA
non-activatable copies (regulatory)
siRNAs (21-25 nt)
Low level transcripts
PTGS
non-activatable copies (silent )
activatable copies (potentially infectious)
Defense against episomal virus
Defense against episomal virus
Epigeneticmodifications
viral suppressor?
terminal redundant transcripts
Transcript level sufficient for activation and
suppressor production
Epigeneticmodifications Epigenetic
modifications
TGS
more transcripts
Weakening of epigenetic control
Virus replicationCell-to-cell spread
Symptoms of infection
Staginnus C, Richert-Pöggeler KR (2006). Endogenous pararetroviruses: two-faced travelers in the plant genome. Trends Plant Sci 11: 485-491.
PVCV may be induced by applying abiotic stress, leading to the development of viral symptoms and increased transcript and siRNA levels.
Molecular cytogenetics lab
Niaz Ali
Pat Heslop-Harrisonts32 @le.ac.uk
www.molcyt.comUserID/PW ‘visitor’
@Pathh1
Rubar Salih
Katja Richert-Poeggeler
Richard Gornall
Thomas Cremer
From cell theory to chromosome theory
Scientific realization and theory alterations in early cell and heredity research
1985
Mendel’s life Mendel’s experiments Why was he forgotten? What was known about chromosomes
at the time of Mendel Mendel’s rediscovery How has chromosome biology
developed since Some examples from our own
research
Overview
150th anniversary
Versuche über Pflanzenhybriden(Experiments with plant hybrids)1865
Gregor Mendel (1822-1884)
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