Examples of use of in situ hybridization in plants for phylogeny and plant breeding

1. Trude Schwarzacher
Department of Biology
University of Leicester, UK
ts32@le.ac.uk
www.molcyt.com

2. Background and history
 What is it?
 What can it do?
 Applications
 Examples from our lab
 Wheat
 Brassica
 Crocus

3. 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
Can be combined with other probes,
most often repetitive DNA probes
 Alien chromatin in breeding lines
 Size and origin
 Recipient chromosome

4. Parental origin of hybrids
 Auto or allo-poliploidy
 Alien chromatin in breeding lines
 Size and origin
 Recipient chromosome
Meiosis and chromosome pairing
 Interphase cytogenetics

6. 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.

8.  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

9. Savidge (1960): allo-tetraploid origin from C. stagnalis and
C. cophocarpa (both 2n=10)
Schotsman (1967):
auto-tetraploid from
C. cophocarpa
C. stagnalis
British Species
Gornall, Johnson and Schwarzacher 2004

12. www.CrocusBank.org

13. Saffron
Crocus sativus
2n=3x=24
C. cartwrightianus green
C. thomasii red

14. Triticeae (Hordeae)
Small grain cereals in the family
of Poaceae (grasses)
wheat
barley
rye
Einkorn
Spelt
wheat Leymus

15. Triticeae
phylogeny

16. Genome size
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;
Rye, 2n=14
Secale cereale

17. Polyploidy
• Polyploids have three or more complete
sets of chromosomes in somatic and
germline cells
triploid 2n=3x
tetraploid 2n=4x
diploid 2n=2x
• Two types of polyploidy
hexaploid
2n=6x
octoploid 2n=8x
Autopolyploidy Allopolyploidy
A x A
AA
AAAA
A x B
AB
AABB

18. Allopolyploids are hybrids
Two 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

19. Wheat evolution and hybrids
Triticum uratu
2n=2x=14
AA
Einkorn
Aegilops speltoides
relative
2n=2x=14
BB
Triticum dicoccoides
Triticum monococcum
2n=2x=14
AA
Bread wheat
Triticum aestivum
2n=6x=42
AABBDD
Durum/Spaghetti
Triticum turgidum ssp durum
2n=4x=28
AABB
2n=4x=28
AABB
Aegilops squarrosa
2n=2x=14
DD
Triticale
xTriticosecale
2n=6x=42
AABBRR
Rye
Secale cereale
2n=2x=14
RR

20. Polyploidy
More than two homologous chromosomes
Diploid: 2x, AA
Triploid: 3x, AAA
Tetraploid: 4x, AAAA
Multivalent formation
2 chromosomes: bivalent
3 chromosomes: trivalent
4 chromosomes: quadrivalent

21. NOR: rDNA loci vary in number,
position and size
Chromosomal
Satellite

23. Triticum aestivum
Bread Wheat
hexaploid wheat
• 2n=6x=42
• three genomes
• AABBDD
• 7 chromosome pairs
each
• homologous 1A, 1A
• homoeologous 1A, 1B, 1D
45S rDNA

24. 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

25. Aneuploidy and chromosome
rearrangements
Additions
Monosomic 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

26. Chromosome engineering:
1BL-1RS wheat translocation
Total genomic rye DNA shows rye-origin
chromosomes
Very common in
Northern Europe and
Canada
Hardier
Better disease
resistance
But loss of bread
making, so used for
feed or biscuit wheat

27. Wheat 1B
Crossa et al 2007 CYMMIYT
Dart markers to link rust and mildew resistance
Wheat proteins: glutenins
on group 1 chromosome
1A, 1B and 1D
Castilho Miller Heslop-Harrison 1996. Physical mapping of
translocation breakpoints in a set of wheat- Aegilops
umbellulata recombinant lines using in situ hybridization.
Theoretical and Applied Genetics 93: 816-825.

30. 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

31. Rye DNA
pTa71-45S rDNA
4 major sites 1RS, 6BS
6 major sites 1RS, 1BS, 6BS
1BL.1RS
1DL.1RS

32. rye chromosome derivative 1R substitutes wheat chromosome 1D
DAPI Rye genomic DNA probe pTa71 (45s rDNA probe)
Forsström and Schwarzacher 2000

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

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

35. Characterization of new sources of
Wheat streak mosaic virus resistance
WSMV resistant and susceptible lines in field
trials
Graybosch RA, Peterson CJ, Baenziger
PS, Baltensperger PD, Nelson LA, Kolmer J, Seaborn B, French R, Hein Martin TJ, Beecher B, Schwarzacher Heslop-Harrison P. 2009. Registration 'Mace' hard red winter wheat. Journal
of Plant Registrations 3(1): 51-56.10.3198/jpr2008.06.0345crc
Six populations of wheat lines that include an alien
chromosome arm from Thinopyrum intermedium carrying
WSMV resistance (Wsm-1 gene)
Bob Graybosch, USDA-ARS, University of Nebraska, USA 6

36. Rapeseed 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)
Genome Specificity of a CACTA (En/Spm) Transposon
Pat Heslop-
Harrison
Karine Alix
Xianhong Ge

37. O. violaceus genomic DNA
DAPI
Xianghong Ge, Farah Badakshi, Heslop-Harrison and Schwarzacher 2010

38.  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

40. Triticeae genomes and
chromosomes
2n = 14 Secale cereale , rye
7 chromosome pairs R genome
1 2 3 4 5 6 7

41. 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)

42. 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

43. Telomere (TTTAGGG)n
Universal in eukaryotes with only a few exceptions
Dynamic
Number of repeats varies: tissue, age and chromosome
Added by telomerase

44. Rye, subteloemreic sequences

45. Differences between genomes
Major differences in the nature and
amount of repetitive DNA
• pTa71
• dpTa1

46. Do repeats reflect genome
relationships and evolution?
pSc119.2
120bp
repeat unit
family
rye
wheat
B genome
dpTa1
Afa-family
340bp
repeat unit
family
wheat
D genome

47. 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
Contento, Heslop-Harrison, Schwarzacher (2005) Cyt. Gen. Res. 109, 34-42

48. S.vav42!248
0.1
Afacer1
T.mono106/42!133pt1
T.mono106/42!155pt1
Ae.umb106/208!1911
Ae.umb106/208!1810
Ae.umb106/208!2012
S.vav147!259pt1
Pet w 25/208!088
Pet w 25/208!077
S.vav106/208!215pt1
S.vav106/208!204pt2
S.vav106/208!215pt2
S.vav25/208!182
S.mon42!136
Pet 22594 25/42!3324
Pet 22594 25/42!3425
CS/325/208!1820
Pet 2259425!315
S.vav25/208!193
T.tau25/147!2524pt2
CS/325/147!2322pt2
L.moll25/42!156
Pet w25/147!123
CS/325/208!1719
CS/325/147!2120
Pet w25/147!3231
Ae.umb25/147!124
981
Ae.umb25/208!168
L.moll25/42!167
119Repeat2
Pet w 25/208!099
S.mon147!1811
S.vav106/208!204pt1
119Repeat3
S.vav147!2711pt1
S.mon25/147!081
T.mono25/208!1212
S.vav42!237pt1
S.vav42!237pt2
T.tau25/147!2625
T.tau25/147!2726
Ae.umb25/208!157
T.tau25/208!1517
T.tau106/42!177
Ae.umb25/42!091
L.moll25/42!189
CS/325/208!1618
T.tau25/147!2524pt1
CS/325/147!2322pt1
Pet 22594 25/208!2325
Pet 22594 25/147!1918
Pet 22594 25/208!2426
T.tau25/208!1315
T.tau25/208!1416
Ae.umb25/147!135
Ae.umb25/147!146
Ae.umb25/208!179
749
1000
996
1000
725
985
564
1000
580
646
942
616
882
998
623
537
915
578
120bp repeat
unit family
in Triticum,
Aegilops and
Secale
species
119Repeat1
Homology between
sequences
70-100%
No species specific
groups
T.tauschii (D
genome)
Irrespective of copy
number in the genome
S. cereale (R genome)

49. het
eu
het
het
het
eu
eu
eu
Chromatin
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

50. Tandem
Repeats

52. 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

53. A
B
C
DNA sequence
Centromere
T
E
Tandem repeat monomer
T
E
Transposable element
Single copy
DNA
Kinetochore
Spindle microtubules pulling
apart chromatids
Metaphase
chromosome
147bp plus 5-70bp linker = 150-220bp
100bp plus 55bp linker = 155bp
D
E
F
G
H
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