Trude Schwarzacher

Department of Biology

University of Leicester, UK

ts32@le.ac.uk

www.molcyt.com

Background and history

What is it?

What can it do?

Applications

Examples from our lab

Wheat

Brassica

Crocus

Uses total genomic DNA as a probe

for in situ hybridization to

chromosomes and nuclei

Identifies origin of chromatin

Parental origin of hybrids

Auto or allo-polyploidy

Alien chromatin in breeding lines

Size and origin

Recipient chromosome

Can be combined with other probes,

most often repetitive DNA probes

Parental origin of hybrids

Auto or allo-poliploidy

Alien chromatin in breeding lines

Size and origin

Recipient chromosome

Meiosis and chromosome pairing

Interphase cytogenetics

Addition of unlabelled,

cold total genomic DNA

to block common

sequences between the

parental genomes

Heslop-Harrison, J.S., Schwarzacher,

T., Leitch, A.R., Anamthawat-Jonsson,

K. and Bennett, M.D. (1988) A method of identifying DNA sequences in

chromosomes of plants. European Patent

Application 8828130.8. December 8, 1988.

Early Publication, June 8, 1990.

Anamthawat-Jonsson, K.,

Schwarzacher, T., Leitch, A.R.,

Bennett, M.D. and Heslop-Harrison,

J.S. (1990) Discrimination between closely related

Triticeae species using genomic DNA as a

probe. Theoretical and Applied Genetics 79,

721-728.

Triticae

Triticum, Aegilops, Hordeum, Haynaldia, Thinopyrum,

Secale, Hystrix, Leymus, Agropyron, Elymus, Elytrigia

Brachypodium

Oryza genomes

Zea mays

Pennesitum

Tripsacum

Saccharum

Avena

Lolium and Festuca hybrids Festulpia

Eleusine

Alstroemeria

Aloe

Lilium

Allium

Crocus

Tulipa

Musa genomes

Asteraceae

Dahlia

Chrysanthemum, Dendranthema and

Argyranthemum

Brassicaceae

Brassica species, alien and hybrids with

Eruca, Orchyophragmus, Sinapis, Raphanus

Brassica, Lesquerella fendleri, Arabidopsis

species/hybrids

Solanaceae

Solanum, potato, tomato

Fabaceae

Arachis

Medicago

Coffea arabica

Gossypium

Rubus

Beta

Zingeria

Setaria

Phalaenopsis

Savidge (1960): allo-tetraploid origin from C. stagnalis and

C. cophocarpa (both 2n=10)

Schotsman (1967):

auto-tetraploid from

C. cophocarpa

C. stagnalis

Gornall, Johnson and Schwarzacher 2004

British Species

www.CrocusBank.org

SaffronCrocus sativus2n=3x=24

C. cartwrightianus green

C. thomasii red

Triticeae (Hordeae)

Small grain cereals in the family

of Poaceae (grasses)

wheat

barley

rye

Einkorn

Spelt

wheat Leymus

Triticeae phylogeny

Genome size

Rye, 2n=14Secale cereale

Hordeum vulgaris (barley) 2x=14 5,550Mb

Secale cereale (rye) 2x=14 8,280Mb

Triticum monoccocum 2x=14 6,230Mb

Aegilops tauschii 2x=14 5,010Mb

Ae. speltoides 2x=14 5,800Mb

T. durum (Durum wheat) 4x=28 12,030Mb

T. aestivum (bread wheat) 6x=42 17,330Mb

Human 3300Mb; Arabidopsis: 180Mb;

• Polyploids have three or more complete sets of chromosomes in somatic and germline cells

• Two types of polyploidy

Polyploidy

triploid 2n=3x

tetraploid 2n=4xdiploid 2n=2x

hexaploid 2n=6x

octoploid 2n=8x

Autopolyploidy Allopolyploidy

A x A

AA

AAAA

A x B

AB

AABB

Allopolyploids are hybridsTwo different genomes: A and B

No or little pairing

Sterile

Allopolyploidy: AABB, AAB, AABBDD

Many plant species have evolved via allopolyploidy

Polyploidy less common in animals

Wheat evolution and hybridsTriticum uratu

2n=2x=14AA

EinkornTriticum monococcum

2n=2x=14AA

Bread wheatTriticum aestivum

2n=6x=42AABBDD

Durum/SpaghettiTriticum turgidum ssp durum

2n=4x=28AABB

Triticum dicoccoides2n=4x=28

AABB

Aegilops speltoidesrelative

2n=2x=14BB

Aegilops squarrosa2n=2x=14

DD

TriticalexTriticosecale

2n=6x=42AABBRR

RyeSecale cereale

2n=2x=14RR

PolyploidyMore than two homologous chromosomes

Diploid: 2x, AA

Triploid: 3x, AAA

Tetraploid: 4x, AAAA

Multivalent formation

2 chromosomes: bivalent

3 chromosomes: trivalent

4 chromosomes: quadrivalent

Chromosomal

Satellite

NOR: rDNA loci vary in number, position and size

Triticum aestivumBread Wheathexaploid wheat• 2n=6x=42

• three genomes• AABBDD• 7 chromosome pairs each• homologous 1A, 1A

• homoeologous 1A, 1B, 1D

45S rDNA

Wheat: normally 2n=6x=42

Chromosome rearrangements or character

Nullisomic: not present

Monosomic: present in one copy

Disomic: present in two copies

Trisomic: present in three copies

Tetrasomic: present in four copies

Aneuploidy and chromosome rearrangements

AdditionsMonosomic addition (not stable)

Plant: 2n = 42+1

Gametes: n = 21 or 21+1

Zygote: 2n = 42, 42+1, 42+2

Disomic addition (more stable)Plant: 2n = 42+2

Gametes: n = 21+1

Substitutions (exchange of chromosomes)Deletions (Chromosome is missing (nulli)

Nulli/Tetra lines

Chromosome engineering:1BL-1RS wheat translocation

Total genomic rye DNA shows rye-origin chromosomes

Very common in Northern Europe and Canada HardierBetter disease resistanceBut loss of bread making, so used for feed or biscuit wheat

Wheat 1B

Crossa et al 2007 CYMMIYTDart markers to link rust and mildew resistance

Wheat proteins: gluteninson group 1 chromosome1A, 1B and 1D

Castilho Miller Heslop-Harrison 1996. Physical mapping of translocation breakpoints in a set of wheat- Aegilopsumbellulata recombinant lines using in situ hybridization. Theoretical and Applied Genetics 93: 816-825.

Total genomic DNA can be

used as a probe to distinguish

Genomes in

sexual hybrids

Alien

chromosome introgression

Additions

Translocations

Used extensively in breeding

programmes to introduce desirable

traits from wild species

Rye DNA

pTa71-45S rDNA

4 major sites 1RS, 6BS

6 major sites 1RS, 1BS, 6BS

1BL.1RS

1DL.1RS

rye chromosome derivative 1R substitutes wheat chromosome 1D

DAPI Rye genomic DNA probe pTa71 (45s rDNA probe)

Forsström and Schwarzacher 2000

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

The genusThynopyrum, including wild goat grasses and wheat grasses, has proven an excellent source for disease and biotic stress resistance

Six populations of wheat lines that include an alienchromosome arm from Thinopyrum intermedium carryingWSMV 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, USA 6

Graybosch RA, Peterson CJ, BaenzigerPS, Baltensperger PD, Nelson LA, JinKolmer J, Seaborn B, French R, Hein G, Martin TJ, Beecher B, Schwarzacher T, Heslop-Harrison P. 2009. Registration of 'Mace' hard red winter wheat. Journal of Plant Registrations 3(1): 51-56.doi: 10.3198/jpr2008.06.0345crc

Rapeseed B. napus (AACC, 2n=4x=38) – hybridized

with C-genome CACTA element redB. oleracea (CC, 2n=2x=18) B. rapa (AA, 2n=2x=20)

Genome Specificity of a CACTA (En/Spm) Transposon

Pat Heslop-

Harrison

Karine Alix

Xianhong Ge

O. violaceus genomic DNADAPI

Xianghong Ge, Farah Badakshi, Heslop-Harrison and Schwarzacher 2010

Parental origin of hybrids Auto or allo-poliploidy

Alien chromatin in breeding lines Size and origin

Recipient chromosome

Meiosis and chromosome pairing

Interphase cytogenetics

Understanding hybrid genomes Chromosome behaviour Chromatin function

Triticeae genomes and chromosomes

1 2 3 4 5 6 7

2n = 14 Secale cereale, rye7 chromosome pairs R genome

120 bp repeat unit family our CS13 probe

- Found in large heterochromatic blocks in rye (Bedbrook et al. Cell, 1980)

- Characterized as pSc119.2 tandem repeat, made of three adjacent subunits (McIntyre et al. Genome, 1990)

- Prevalence in Triticeae (Hordeae) species

- Found in the sister tribe Aveneae

present in many wheat genomes (A, B, R, AB, ABR)

TAS (Telomeric Associated Sequences)

Location close to telomeric repeats in rye and wheat/barley

Wheat/Barley

pSc119.2

(TTTAGGG)n

Mao et al. Mol Gen Genet

1997

Rye

pSc119.2 pSc250 pSc200 (TTTAGGG)n

Vershinin, Schwarzacher and Heslop-

Harrison. Plant Cell 1995

• Telomeric sites (telomeric repeats and TASs) are

more variable than any other region of plant

genome

• High sequence variation of 120 bp family

members within each genome in diploid and

polyploid Triticeae species

Telomere (TTTAGGG)n

Universal in eukaryotes with only a few exceptions

Dynamic

Number of repeats varies: tissue, age and chromosome

Added by telomerase

Rye, subteloemreic sequences

• pTa71

Differences between genomesMajor differences in the nature and amount of repetitive DNA

• dpTa1

Do repeats reflect genome

relationships and evolution?pSc119.2

120bp repeat unit family

rye wheatB genome

dpTa1

Afa-family

340bp repeat unit familywheat D genome

Contento, Heslop-Harrison, Schwarzacher (2005) Cyt. Gen. Res. 109, 34-42

120-bp repeat unit family: characteristics

• High sequence variation of family members (75 clones)

• Few insertions and deletions, but mainly single nucleotide mutations keep the

repeat a constant length

• Characteristics with telomere-associated sequences (TASs)

- High sequence homology to TASs from wheat (PSR2151, Mao et al 1997) and barley

(HTV02, Belostotsky et al.1989)

- Plant telomere-similar sequences: AAAACCCC or AAAACCGG

- Conserved imperfect inverted repeat (palindrome) of 20 bp: A2CGCAC4G4T2CGT2

S.vav42!248

0.1

Afacer1T.mono106/42!133pt1T.mono106/42!155pt1

Ae.umb106/208!1911Ae.umb106/208!1810Ae.umb106/208!2012

S.vav147!259pt1Pet w 25/208!088Pet w 25/208!077

S.vav106/208!215pt1S.vav106/208!204pt2

S.vav106/208!215pt2S.vav25/208!182

S.mon42!136Pet 22594 25/42!3324

Pet 22594 25/42!3425CS/325/208!1820

Pet 2259425!315S.vav25/208!193

T.tau25/147!2524pt2CS/325/147!2322pt2

L.moll25/42!156Pet w25/147!123

CS/325/208!1719CS/325/147!2120

Pet w25/147!3231Ae.umb25/147!124

Ae.umb25/208!168L.moll25/42!167

119Repeat2Pet w 25/208!099

S.mon147!1811S.vav106/208!204pt1

119Repeat3S.vav147!2711pt1S.mon25/147!081

T.mono25/208!1212

S.vav42!237pt1S.vav42!237pt2

T.tau25/147!2625T.tau25/147!2726

Ae.umb25/208!157T.tau25/208!1517

T.tau106/42!177Ae.umb25/42!091

L.moll25/42!189CS/325/208!1618

T.tau25/147!2524pt1CS/325/147!2322pt1

Pet 22594 25/208!2325Pet 22594 25/147!1918

Pet 22594 25/208!2426T.tau25/208!1315

T.tau25/208!1416Ae.umb25/147!135

Ae.umb25/147!146Ae.umb25/208!179

7491000

996

1000

725

985

564

1000

580

646

942

616

882

998

981

623

537

915

578

120bp repeat unit familyin Triticum, Aegilops and Secalespecies

119Repeat1

Homology between sequences

70-100%

No species specific groups

Irrespective of copy

number in the genome

T.tauschii (D genome)

S. cereale (R genome)

euchromatin is lightly stained, is generally gene rich, less ‘condensed’ and more transcriptionally active

heterochromatin is strongly stained, is highly ‘condensed’ and relatively deficient in genes and those present often show decreased transcriptional activity; rich in repetitive DNA sequences

het

het

het

het

eueu

eu

eu

Chromatin

Tandem Repeats

DNA packs around nucleosomes

Linker: 0 – 20bp

Coverage for one nucleosome: 160-180bp

two nucelosomes: 320-360bp

3 units of 120bp cover two nucleosomes

Many repeats fit around

the nucleosomes

Arabidopsis 180bp repeat

pSc119.2: 120bp

Afa family: 340bp

A

B

C

Centromere

DNA sequence

T

E

Tandem repeat monomer

T

E

Transposable element

Single copy

DNA

Spindle microtubules pulling

apart chromatids

Metaphase

chromosome

147bp plus 5-70bp linker = 150-220bp

100bp plus 55bp linker = 155bp

D

E

F

G

H

Kinetochore

Heslop-Harrison JS, Schwarzacher T. 2013. Nucleosomes and centromeric DNA packaging. Proc Nat Acad Sci

USA. http://dx.doi.org/10.1073/pnas.1319945110. See also http://wp.me/p2Ewqp-7h