Plant Chromosomes: European Cytogeneticists outline: Trude Schwarzacher and Pat Heslop-Harrison

Plant Molecular Cytogenetics Trude Schwarzacher [email_address] www.molcyt.com Including talk slides! www.molecularcytogenetics.com UserID/PW ‘visitor’ Nimes ECA Course 27 February 2012

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

‘ Golden Yellow’ triploid 2n=3x=14 C. flavus 2n=2x=8 (8 yellow) C. angustifolius 2n=2x=12 (6 green) Orgaard, Jacobsen & HH ‘ Stellaris’ hybrid diploid 2n=2x=10 C. 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 Cytogenetics
Plant chromosomes
Plant genomes and their sizes
DNA components of a chromosome
Retroelements
Chromosomes and sources of genetic markers
Chromosome and genome painting
Polyploids and cereal chromosome evolution
Plant breeding and aliens
Part I: Part II:

Somatic metaphase chromosomes Arabidopsis Human Pine Centromere Telomere

Darwin: The final paragraph of “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.

OBJECTIVES Fundamental and Practical
Explain major structures and features of the DNA in plant genomes
Understand the structure of chromosomes and genomes
Explain the nature and origin of molecular markers
Understand 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 species
Decide 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 farmers
Use the literature relating to genomics, genetics and plant breeding and communicate it in writing

The Genome and Chromosomes
The DNA is organized in multiple chromosomes
Each is a single, linear DNA molecule
The DNA is packaged around proteins (histones)
The nuclear chromosome has special sequences at its ends
There are separate genomes in the plastids and mitochondria
There 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 Mbp Sugar beet Beta vulgaris 63% 758 Mbp Broad bean Vicia faba 85% 12000 Mbp Rye Secale cereale 92% 8800 Mbp Onion 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 suecica Hybrid 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 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 genome Heslop-Harrison & Schmidt 2012. Encyclopedia of Life Sciences Other genes

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

Oil Palm
32 chromosomes DAPI;
TTTAGGG telomere;
45S rDNA (1 major pair + minor)
5S rDNA (1 major + minor)
02/07/12

Repetitive Sequences
The majority of the genomic DNA in most species (95% sometimes)
Tandem Repeats
Simple Sequence Repeats
Dispersed Repeats
Functional Repeats
Retroelements

Retrotransposons Class I transposable elements RNA intermediate DNA transposons Class 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 red B. oleracea (CC, 2n=2x=18) B. rapa (AA, 2n=2x=20) Alix & HH 2008

Triticale: wheat x rye hybrid

Total genomic DNA can be used as a probe to distinguish
Genomes in sexual hybrids
Alien chromosome introgression

dpTa1 Aegilops ventricosa DDNN ABDN AABBDDNN Marne AABBDD CWW1176-4 Rendezvous Piko VPM1 Dwarf A 96ST61 Virtue × × × × Hobbit × {Kraka × (Huntsman × Fruhgold)} Triticum persicum Ac.1510 AABB Inheritance of Chromosome 5D

rRNA gene expression in Triticale
Four expression sites Six gene sites

Modification of DNA Methylation
Methylation widely implicated in gene expression control
Treat with 5-azacytidine
N at carbon-5 position not C so –CH 3 cannot be added
Effect of treatment on Triticale
Ag-NOR method
see 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 5SrRNA Nor-1 (d) Em Gli-1 Tri-1 Physical map vs Genetic map Genes are often clustered Genes (and recombination) are often distal

Wsm-1: only effective source of resistance to WSMV

dpTa1 digoxigenin IWG genomic DNA biotin

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

Retroelement Markers
Insertion IRAP – InterRetroelement PCR Retrotransposon LTR LTR Retrotransposon LTR LTR Retrotransposon LTR LTR Retrotransposon LTR LTR Retrotransposon LTR LTR Retrotransposon LTR LTR

IRAP diversity in Musa Teo, Tan, Ho, Faridah, Othman, HH, Kalendar, Schulman 2005 J Plant Biol Nair, Teo, Schwarzacher, HH 2006 Euphytica Desai, Maha … , HH et al. in prep.

07/02/12 TCCCTGAG: 8-bp TSD. 30-bp TIRs 273-bp hAT ACCCACCTGGCTCTTGTGTC ATACCATTGAAAAGCCGATTATATTTGTCCCCATTCATCCAAAAGA TCCCTGAG CAAGGTCTG C CATACCG T A C CGTACCG G CG TTTCGAC CCGG GCTCGGTACGGTA CCGG TGTA CCGG GCAGTACATCAGGGTGTACCGAATGGTACACCCTGATGTACCGAACAATTTTATACTTTTTCATACTGTAGCAGTGCTACAGTATAATACTGTAGCACTGTAGCGGTATCGGGCGGTCCGCGTA CCGG TAACCTGTCGGA CCGG TACATACCGC CCGG TAT CG G CGGTACG C T T CGGTATG A CAGACCTTG TCCCTGAG TATATATCTCTTTTCTAAATTTATGACCACTCCAAGGCAACTTGCCAAAGAAAATGAAAAGAAGAAAAAAATTAGGGGAATGAAGATTCTCCACAATTCCTTATTCTTTGATTTGAGATAAATAATGTCCATAGTAAAACATATCTTATGATCATCATTGCTGATTAATCAAAATACCTGATTCTATAGTCTCAAGCTTT AGTGGTCAAAACACATTCGC TSD TIR TIR TSD hAT1 in Musa acuminata F and R primers indicated by blue arrows in sequence Musa balbisiana Musa acuminata a) b)

07/02/12 hAT1 insertion sites in Musa diversity collection hAT486F and hAT037R Top bands (560-bp) amplified hAT element and lower bands amplifying the flanking sequences only 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 1KB 800 600 400 200

Nuclear Copies of Banana Streak Virus in Banana

DNA Fibre Hybridization

Evolution  Epigenetics  Development
Phenotype
Multiple abnormalities
Genetic changes
non-reverting
Changes seen, some reverting
(Male/Female)
Normal Differentiation
Cause
Chromosomal loss, deletion or translocation
Gene mutation / base pair changes
Telomere shortening
Retro)transposon insertion
Retrotransposon activation
SSR expansion
Methylation
Heterochromatinization
Chromatin remodelling
Histone modification

From Chromosome to Nucleus Pat Heslop-Harrison [email_address] www.molcyt.com

United Nations Millennium Development Goals- MDGs
Goal 1 – Eradicate extreme poverty and hunger
Goal 2 – Achieve universal primary education
Goal 3 – Promote gender equity and empower women
Goal 4 – Reduce child mortality
Goal 5 – Improve maternal health
Goal 6- Combat HIV/AIDS, malaria and other diseases
Goal 7 - Ensure environmental sustainability
Goal 8 - Develop a global partnership 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

Plant Molecular Cytogenetics Trude Schwarzacher [email_address] Website: www.molcyt.com or www.molecularcytogenetics.com UserID/PW ‘visitor’ To download full text of papers Nimes ECA Course 27 February 2012

Plant Chromosomes: European Cytogeneticists outline: Trude Schwarzacher and Pat Heslop-Harrison

by Pat Heslop-Harrison

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