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)