In situ hybridization results and examples for course Trude Schwarzacher
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Transcript of In situ hybridization results and examples for course Trude Schwarzacher
Trude Schwarzacher
Department of Biology
University of Leicester, 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