Nimes ECA Course27 February 2012

Plant Molecular Cytogenetics

Trude Schwarzacherts32@le.ac.uk

www.molcyt.comIncluding talk slides!

www.molecularcytogenetics.comUserID/PW ‘visitor’

The most popular garden varieties are sterile, diploid or triploid interspecific hybrids

‘Golden Yellow’ triploid 2n=3x=14C. flavus 2n=2x=8 (8 yellow) C. angustifolius 2n=2x=12 (6 green)

Orgaard, Jacobsen & HH

‘Stellaris’ hybrid diploid 2n=2x=10C. flavus 2n=2x=8 (4 green)

C. angustifolius 2n=2x=12 (6 blue)

Metaphase I in triploid Golden Yellow Crocus

In many metaphase I cells of this triploid, we see four bivalents from pairing of the four pairs of C. flavus (2n=2x=8)-origin chromosomes, with the six chromosomes from C. angustifolius (2n=2x=12) present as univalents. Alternative configurations also seen.

Antibodies below to meiosis components

Plant genome sizes vary over a 2350-fold range

Plant Molecular CytogeneticsPlant chromosomesPlant genomes and their sizesDNA components of a chromosomeRetroelementsChromosomes and sources of genetic markers

Chromosome and genome paintingPolyploids and cereal chromosome evolutionPlant breeding and aliens

Part I:

Part II:

Centromere Telomere

Somatic metaphase chromosomes

Arabidopsis Human Pine

Darwin: The final paragraphof “The Origin”

It is interesting to contemplate … many plants of many kinds … and to reflect that these elaborately constructed forms, so different from each other

… .have all been produced by laws acting around us … from so simple a beginning endless forms most beautiful and most wonderful have been, and are being evolved.

OBJECTIVESFundamental and Practical

Explain major structures and features of the DNA in plant genomesUnderstand the structure of chromosomes and genomesExplain the nature and origin of molecular markersUnderstand key events in evolution and generation of diversity including induced mutations

OBJECTIVES Fundamental and Practical

 Understand how genomes and diversity can be manipulated and exploited Relate genome information and models to the applications in the genome of particular speciesDecide which molecular markers are appropriate for various applications Introduce the concept of superdomestication into breeding programmes and consider solutions to major problems facing breeders and farmersUse the literature relating to genomics, genetics and plant breeding and communicate it in writing

The Genome and ChromosomesThe DNA is organized in multiple chromosomesEach is a single, linear DNA moleculeThe DNA is packaged around proteins (histones)The nuclear chromosome has special sequences at its ends

There are separate genomes in the plastids and mitochondriaThere can be viral and bacteria-like genomes in the nucleus and cytoplasm

Genes!

Repetitive DNA-Sequences form the largest part of the genome

Arabidopsis thaliana>25% 145 MbpSugar beet Beta vulgaris 63% 758 MbpBroad bean Vicia faba 85% 12000 MbpRye Secale cereale 92% 8800 MbpOnion Allium cepa 95% 15100 Mbp

These species are all diploid – 2x

Species Repetitive DNA Genome size

Human Homo sapiens 35% 3000 Mbp

Arabidopsis thaliana 2n=10

Arabidopis suecicaHybrid A. arenosa (pAa214 green) X A. thaliana (180bp

red)

Kamm, HH et al.

ancestral

A B C D

High-copy number High-copy number High-copy number High-copy number

E F

Low-copy number Low-copy number

New species-specific variants

High copy spp: homogenized old (ABC) or new (D) variants

Low copy spp: most old variants in low copy number (EF)

See Kuhn, HH et al. 2009. Heredity& 2008. Chr. Res.

Organelle sequences

from chloroplasts or

mitochondriaSequences 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

Derivative chromosome 1R of Lines 7-102 and 7-169Derivative chromosome 1R of Lines 7-102 and 7-169

04/13/23

Oil Palm

32 chromosomes DAPI; TTTAGGG telomere; 45S rDNA (1 major pair + minor)

5S rDNA (1 major + minor)

RepetitiveSequences

The majority of the genomic DNA in most species (95% sometimes)Tandem RepeatsSimple Sequence Repeats Dispersed RepeatsFunctional RepeatsRetroelements

RetrotransposonsClass I transposable elementsRNA intermediate

DNA transposonsClass II transposable elements

Cut-and-paste

Genome Specificity of a CACTA (En/Spm) Transposon

B. napus (AACC, 2n=4x=38) – hybridized with C-genome CACTA element redB. oleracea (CC, 2n=2x=18) B. rapa (AA, 2n=2x=20) Alix & HH 2008

Triticale:wheat x ryehybrid

Total genomic DNA can be used as a probe to distinguish

• Genomes in Genomes in sexual hybridssexual hybrids

• Alien Alien chromosome chromosome introgressionintrogression

dpTa1

Aegilops ventricosaDDNN

ABDN

AABBDDNN MarneAABBDD

CWW1176-4

Rendezvous

Piko

VPM1 Dwarf A

96ST61

Virtue

×

×

×

×

Hobbit

× {Kraka × (Huntsman × Fruhgold)}

Triticum persicum Ac.1510AABB

Inheritance of Inheritance of Chromosome Chromosome

5D5D

rRNA gene expression in Triticale

Four expression sites Six gene sites

Modification of DNA Methylation

Methylation widely implicated in gene expression controlTreat with 5-azacytidine

N at carbon-5 position not C so –CH3 cannot be added

Effect of treatment on Triticale

Ag-NOR methodsee www.methods.molcyt.com methods page

Copyright restrictions may apply.

Saeidi, H. et al. Ann Bot 2008 101:855-861; doi:10.1093/aob/mcn042

UPGMA dendrograms of the relationships based on IRAP analysis of (A) accessions of Ae. tauschii subsp

Adpg2-1

Glu-1

Xpsr161

Xpsr957

Xpsr381

5SrRNANor-1

(d) Em

Gli-1

Tri-1

Physical map vsGenetic map

Genes are often clusteredGenes (and recombination)

are often distal

Wsm-1: only effective source of resistance to Wsm-1: only effective source of resistance to WSMVWSMV

dpTa1 digoxigenin

IWG genomic DNA biotin

Wild banana species:Musa acuminata – A genome Musa balbisiana – B genomeBasic chromosome number n=x=11 Genome size c. 550 MbpMost cultivated hybrids are 2n=3x=33

Retroelement Markers

Retrotransposon LTRLTR

Retrotransposon LTRLTR

RetrotransposonLTR LTR

Retrotransposon LTRLTR

Insertion

IRAP – InterRetroelement PCR

Retrotransposon LTRLTR

RetrotransposonLTR LTR

IRAP diversity in Musa

Teo, Tan, Ho, Faridah, Othman, HH, Kalendar, Schulman 2005 J Plant BiolNair, Teo, Schwarzacher, HH 2006 Euphytica Desai, Maha…, HH et al. in prep.

13/04/23

TCCCTGAG: 8-bp TSD.30-bp TIRs

273-bp hAT

ACCCACCTGGCTCTTGTGTCATACCATTGAAAAGCCGATTATATTTGTCCCCATTCATCCAAAAGATCCCTGAGCAAGGTCTGCCATACCGTACCGTACCGGCGTTTCGACCCGGGCTCGGTACGGTACCGGTGTACCGGGCAGTACATCAGGGTGTACCGAATGGTACACCCTGATGTACCGAACAATTTTATACTTTTTCATACTGTAGCAGTGCTACAGTATAATACTGTAGCACTGTAGCGGTATCGGGCGGTCCGCGTACCGGTAACCTGTCGGACCGGTACATACCGCCCGGTATCGGCGGTACGCTTCGGTATGACAGACCTTGTCCCTGAGTATATATCTCTTTTCTAAATTTATGACCACTCCAAGGCAACTTGCCAAAGAAAATGAAAAGAAGAAAAAAATTAGGGGAATGAAGATTCTCCACAATTCCTTATTCTTTGATTTGAGATAAATAATGTCCATAGTAAAACATATCTTATGATCATCATTGCTGATTAATCAAAATACCTGATTCTATAGTCTCAAGCTTTAGTGGTCAAAACACATTCGC

TSD TIR TIR TSD

hAT1 in Musa acuminataF and R primers indicated by blue arrows in sequence

Musa balbisiana

Mu

sa

acu

min

ata

41

a)

b)

13/04/23 42

HP-1 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 41 42 43 44 45 46 47 48

1KB800600400200

hAT1 insertion sites in Musa diversity collectionhAT486F and hAT037RTop bands (560-bp) amplified hAT element and lower bands amplifying the flanking sequences only

Nuclear Copies of Banana Streak Virus in Banana

DNA Fibre Hybridization

EvolutionEpigeneticsDevelopment

PhenotypeMultiple abnormalities

Genetic changes  non-reverting

Changes seen, some reverting

(Male/Female)Normal Differentiation

CauseChromosomal loss, deletion or

translocationGene mutation / base pair changes

Telomere shorteningRetro)transposon insertion Retrotransposon activation

SSR expansionMethylation

HeterochromatinizationChromatin remodellingHistone modification

From Chromosome to Nucleus

Pat Heslop-Harrison phh4@le.ac.uk www.molcyt.com

United Nations United Nations Millennium Development Goals- MDGsMillennium Development Goals- MDGs

• Goal 1 – Eradicate extreme Goal 1 – Eradicate extreme poverty and hunger poverty and hunger

•Goal 2 – Achieve universal primary educationGoal 2 – Achieve universal primary education

• Goal 3 – Promote gender equity Goal 3 – Promote gender equity and empower womenand empower women

• Goal 4 – Reduce child mortalityGoal 4 – Reduce child mortality• Goal 5 – Improve maternal Goal 5 – Improve maternal

health health • Goal 6- Combat HIV/AIDS, malaria Goal 6- Combat HIV/AIDS, malaria

and other diseasesand other diseases• Goal 7 - Ensure environmental Goal 7 - Ensure environmental

sustainabilitysustainability• Goal 8 - Develop a global Goal 8 - Develop a global

partnership for developmentpartnership for development

Conventional Breeding

Superdomestication

Cross the best with the best and hope for something better

Decide what is wanted and then plan how to get it- variety crosses- mutations- genepool

- genes

50 years of plant breeding progress

CytoGenomics …

The genepool has the diversity to address these challenges …New methods to exploit and characterize germplasm let use make better and sustainable use of the genepool

Nimes ECA Course27 February 2012

Plant Molecular Cytogenetics

Trude Schwarzacherts32@le.ac.uk

Website: www.molcyt.com orwww.molecularcytogenetics.com

UserID/PW ‘visitor’To download full text of papers