The document discusses using cytogenetics and molecular cytogenetics to study polyploidy signatures in crop karyotypes and breeding materials. Recent polyploidy can be revealed through hybridization and cytogenetics, while ancient polyploidy is revealed by sequencing. Understanding polyploidy is important for speciation, evolution and breeding. Different sequences classes evolve at different rates, and molecular cytogenetics can provide insights into recent rearrangements or duplications as well as ancient evolutionary polyploidy. The consequences and applications of studying polyploidy in crops are also discussed.

1. geneticists Association
e 2013 Dublin
Polyploidy: ancient andPolyploidy: ancient and
modern signatures inmodern signatures in
karyotypes of crops andkaryotypes of crops and
breeding materialbreeding material
Pat Heslop-Harrison
Phh4@le.ac.uk
Website: www.molcyt.com or
www.molecularcytogenetics.com
UserID/PW ‘visitor’
To download full text of papers

2. Recent polyploidy
◦ Revealed by cytogenetics and hybridization
Recent rearrangements or duplications
◦ Revealed by molecular cytogenetics
Ancient, evolutionary polyploidy
◦ Revealed by sequencing
Understanding polyploidy is important for
speciation, evolution and breeding
Different sequence classes evolve at
different rates and many are saltatory
rather than clocks
Consequences and applications

3. DasypyrumDasypyrum
breviaristatumbreviaristatum
2n=4x=282n=4x=28
Is itIs it AAAAAAAA oror AAAABB??
D. villosum (genomic
DNA green) × D.
breviaristatum (red)
Meiotic metaphase I in a
F1 hybrid showing
autotetraploid nature
Galasso et al.

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

5. Satellite DNA probe green

7. Evolution of Wheats - PolyploidyEvolution of Wheats - Polyploidy
Common Ancestor
2n=2x=14
Aegilops ventricosa
2n=4x=28
DDNN
Triticum tauschii
2n=2x=14
DD
Aegilops uniaristata
2n=2x=14
NN
Triticum aestivum
2n=6x=42
AABBDD
Triticum durum
2n=4x=28
AABB
Triticum
monococcum
2n=2x=14
AA
Aegilops sp.
2n=2x=14
BB
Aegilops
2n=2x=14
Triticum
2n=2x=14

8. Lodging in cereals

9. Use of repetitiveUse of repetitive
DNA sequences asDNA sequences as
chromosomechromosome
markersmarkers

11. Aegilops tauschiiAegilops tauschii in Iranin Iran
Dpta1
dpTa1-
Repetitive
banding pattern
does correlate
with taxonomic
grouping

12. 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

14. Wild diploid bananaWild diploid banana
Cavendish : ‘Western’
banana cultivar
2n=3x=33;
AAA genomes

16. RetroelementsRetroelements
Sequences which amplify through an RNASequences which amplify through an RNA
intermediateintermediate
50% of all the DNA!

17. Alignment of two BACs shows gaps in both A and B genome
MA4_82I11
MBP_81C12
MuhAT
1
MuhAT2
a
XX TE (SINGLE)XX TE MITE
XX TE
(AGNABI)
MuhAT3 MuhAT4 MITE(MBIR
)
XX TE XX TE (MBT)
272 bp 102,190
bp
26, 410 bp 128,068 bp
DNA transposons hAT are particularly frequent
hAT (named from hobo (Drosophila), Ac-Ds (maize), and Tam3 (Antirrhinum)
transposon are characterized by an 8 bp TSD8 bp TSD, and short TIRs of 5–27 bpshort TIRs of 5–27 bp
flanking a transposase (sometimes degenerate) including a DDE site.
Non-autonomous (sometimes called MITEs, miniature inverted transposable
elements) derivatives of hAT elements are also found, with deletion of most of
the coding sequence, and may amplify to huge copy numbers.
Menzel, Schmidt, Nouroz, HH in prep 2013

18. RetroelementsRetroelements
Homologous BAC sequences from Calcutta
4 Homologous over the full length
except for a 5kb insert
a Ty1-copia retroelement

19. 01/07/13 19
Sr. No. Primer Pairs Product Size
(bp)
Sequence
1. hAT18486
hAT19037
560 ACCCACCTGGCTCTTGTGTC
AGCGAATGTGTTTTGACCAC
MBP 81C12 (M. balbisiana) x MA4 82I11 (M. acuminata) BACs.
Musa balbisiana (MBP 81C12)
Musaacuminata(MA482I11)
Transposed Element
hAT 1
hAT 2
hAT 4
Microsatellite (AT)
hAT 3621 bp MBT
384 bp TE + 781 MITE
1676 TE
Microsatellite (AT)
4192 bp TE

20. 01/07/13 20
Sr. No. Primer Pairs Product Size
(bp)
Sequence
1. hAT18486
hAT19037
560 ACCCACCTGGCTCTTGTGTC
AGCGAATGTGTTTTGACCAC
MBP 81C12 (M. balbisiana) x MA4 82I11 (M. acuminata) BACs.
Musa balbisiana (MBP 81C12)
Musaacuminata(MA482I11)
Transposed Element
hAT 1
hAT 2
hAT 4
Microsatellite (AT)
hAT 3621 bp MBT
384 bp TE + 781 MITE
1676 TE
Microsatellite (AT)
4192 bp TE

21. A-genome specific hAT in three
Musa hybrids (2n=3x=33)
Musa ‘Williams Cavendish’
(AAA)
Musa
(ABB)
Musa
(ABB)

22. 01/07/13 22
Dot plot showing the complete Inverted
repeat.

23. 01/07/13 23
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
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 – Menzel, Nouroz, Schmidt, Schwarzacher, Heslop-Harrison 2013/14

24. Retroelement Markers
Retrotransposon
LTRLTR
Retrotransposon
LTRLTR
RetrotransposonLTR LTR
Retrotransposon
LTRLTR
Insertion
IRAP – InterRetroelement PCR
Retrotransposon
LTRLTR
RetrotransposonLTR LTR

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

26. 01/07/13 26Phylogenetic analysis of Musa genomes – separating species. Teo, Schwarzacher et al.

27. Diploid 2n=2x=22 Musa / banana metaphase probed red with transposable element

29. Timing of LTR Retrotransposon insertion in Musa
Red 0 to 2 Mya, yellow 2 to 4 Mya, green 4 to 6 Mya and blue older
D’Hont et al. 2012

30. The Banana GenomeThe Banana Genome
Seven countries + international organization
coordinated by Angelique D’Hont - France
(CIRAD, Genoscope)

31. 523 Mb DH-Pahang genome size (flow
cytometry)
27.5 million Roche/454 single reads 16 x
coverage
2.1 million Sanger reads 4 x coverage
50.3 x of Illumina data
54 BAC sequences (1%)

32. A D’Hont et al. Nature 000, 1-5 (2012) doi:10.1038/nature11241
Six-way Venn diagram showing the distribution of shared gene
families (sequence clusters) among M. acuminata, P. dactylifera,
Arabidopsis thaliana, Oryza sativa, Sorghum bicolor and Brachypodium
distachyon genomes.

34. A D’Hont et al. Nature 000, 1-5 (2012) doi:10.1038/nature11241
Whole-genome duplication events.

35. A D’Hont et al. Nature 2012
doi:10.1038/nature11241

37. A D’Hont et al. Nature 2012
doi:10.1038/nature11241

38. 1010 µµmm
DNA methylation is unevenly distributed onDNA methylation is unevenly distributed on
MusaMusa chromosomeschromosomes
copiacopia
elementselements
in methylatedin methylated
regions, but alsoregions, but also
in some lowin some low
methylatedmethylated
regions (arrows)regions (arrows)
5MeC

39. 1010 µµmm
C.H Teo and Schwarzacher
5MeC
DNA methylation is unevenly distributed onDNA methylation is unevenly distributed on
MusaMusa chromosomeschromosomes
gypsygypsy
elementselements
in methylatedin methylated
regions, but alsoregions, but also
in some lowin some low
methylatedmethylated
regions (arrows)regions (arrows)
Teo &Teo &
Schwarzacher inSchwarzacher in
prep 2013prep 2013

40. Arachis hypogaeaArachis hypogaea - Peanut- Peanut
Tetraploid of recent origin,Tetraploid of recent origin,
ancestors separated only 3 My agoancestors separated only 3 My ago
Ana Claudia Araujo, David Bertioli, PHH et al.
Embrapa, Brasília. Annals Botany in press 2013

41. A2n=4x=40probed
(A.duranered)A.ipaënsis

Be

42. Arachis duranensis BAC probes on A.
hypogea chromosomes
Right: green BAC AD52G19;red AD79O23
Below: red AD51I17
Below right: green AD179B13;red 5S
rDNA

43. paradox in the evolution of genome
structure: the predominant repetitive DNA
genome fraction is in evolutionary flux,
whilst, at the same time, low copy
number DNA is conserved over
evolutionary time

44. Size and location of
chromosome regions
from radish (Raphanus
sativus) carrying the
fertility restorer Rfk1
gene and transfer to
spring turnip rape
(Brassica rapa)
DAPI metaphase blue
Radish genomic red
(labels 2 radish
chromosomes and 45S
rDNA)
Rfk1 carrying BAC green
labels sites on radish and
homoeologous pair in
Brassica
Tarja Niemelä,
Seppänen, Badakshi,
Rokka HH
Chromosome Research
2012

46. Recent polyploidy
◦ Revealed by cytogenetics and hybridization
Recent rearrangements or duplications
◦ Revealed by molecular cytogenetics
Ancient, evolutionary polyploidy
◦ Revealed by sequencing
Understanding polyploidy is important for
speciation, evolution and breeding
Different sequence classes evolve at
different rates and many are saltatory
rather than clocks
Consequences and applications

47. Anything special about cropAnything special about crop
genomes?genomes?
Crop Genome size 2n Ploidy Food
Rice 400 Mb 24 2 3x endosperm
Wheat 17,000 Mbp 42 6 3x endosperm
Maize 950 Mbp 10 4 (palaeo-tetraploid) 3x endosperm
Potato 900 Mbp 48 4 Modifed leaf
Sugar beet 758 Mbp 18 2 Modified root
Cassava 770 Mbp 36 2 Tuber
Peanut 2,800 Mbp 40 4 Seed cotyledon
Oil palm 3,400 Mbp 32 Fruit mesocarp
Banana 523 Mbp 2x 33 3 Fruit mesocarp
Heslop-Harrison 2012. www.tinyurl.com/domest

48. 50 years of plant breeding50 years of plant breeding
progressprogress
Global production figuresGlobal production figures
Agronomy&
Nitrogen
Genetics
Transgenic
Bt insect-resistant maize
(& herbicide tolerant)

49. CytoGenomics …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

50. www.molcyt.comwww.molcyt.com
phh4@le.ac.ukphh4@le.ac.uk
Recent polyploidy
◦ Revealed by cytogenetics and hybridization
Recent rearrangements or duplications
◦ Revealed by molecular cytogenetics
Ancient, evolutionary polyploidy
◦ Revealed by sequencing
Understanding polyploidy is important for
speciation, evolution and breeding
Different sequence classes evolve at
different rates and many are saltatory
rather than clocks
Consequences and applications

51. geneticists Association
e 2013 Dublin
Polyploidy: ancient andPolyploidy: ancient and
modern signatures inmodern signatures in
karyotypes of crops andkaryotypes of crops and
breeding materialbreeding material
Pat Heslop-Harrison
Phh4@le.ac.uk
Website: www.molcyt.com or
www.molecularcytogenetics.com
UserID/PW ‘visitor’
To download full text of papers