Plant Molecular Cytogenetics www.molcyt.com Conference Katowice, Poland September 2014 Chromosomes, in situ hybridization, genome organization and evolution

1. Chromosomes, Crops and
Superdomestication in Katowice
Pat Heslop-Harrison
phh4@le.ac.uk
www.molcyt.com
UserID/PW ‘visitor’
Pathh1:
Twitter #PMC .
Slideshare pathh1

2. From Chromosome to Nucleus
Pat Heslop-Harrison phh4@le.ac.uk www.molcyt.com

3. How do genomes evolve?
–Gene mutation Genome very rarely evolution
(human:
10−8/site/generation)
–Chromosome evolution
–Polyploidy and genome duplication (ancient &
modern)
–Repetitive sequences: mobility & copy number
(10−4/generation in μsat)
–Recombination
–Epigenetic aspects: centromeres & expression

4. How do genomes evolve?
– Gene mutation Genome very rarely
evolution
– Chromosome evolution
– Polyploidy and genome duplication (ancient and modern)
– Repetitive sequences: mobility & copy number
– Recombination
– Epigenetic aspects – centromeres & expression
How can we exploit knowledge of genome evolution?
– Biodiversity
– Chromosome and genome engineering
– Breeding
– Markers

6. Musa biodiversity and genomes: x=11
Red - AAA 2n=3x=33 – M. acuminata
Palayam codan AAB (two bunch yellow, one green) Musa x
Peyan ABB (green cooking banana)
Njalipoovan AB (yellow) 2n=2x=22 M. acuminata x M. balbisiana
Robusta AAA (green ripe)
Nendran AAB
Poovan AAB (one yellow bunch)
Red AAA
Varkala, Kerala, India Peyan ABB

8. Retrotransposons
Class I transposable elements
RNA intermediate
DNA transposons
Class II transposable elements
Cut-and-paste

9. Retroelements
Sequences which amplify through an RNA intermediate
• 50% of all the DNA!

10. Retroelements
BAC sequences from Musa Calcutta 4
Homologous over the full length
except for a 5kb insert
• a Ty1-copia retroelement

11. Alignment of two homologous Musa BACs shows gaps in both
B genome M. balbisiana and A genome M. acuminata
MA4_82I11
MBP_81C12
MuhAT
1
XX TE MITE XX TE (SINGLE)
MuhAT2
a
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
8 bp TSD, and short TIRs of 5–27 bp
transposase (sometimes degenerate) including a DDE site.
Non-autonomous (MITE) derivatives of hAT with deletion coding sequence
Menzel, Schmidt, Nouroz, HH Chr Res subject minor revision 2015

12. Musa balbisiana (MBP 81C12)
Musa acuminata (MA4 82I11)
hAT 1
1676 TE
384 bp TE + 781 MITE
Transposed Element
Sr. No. Primer Pairs Product Size
(bp)
Microsatellite (AT)
621 bp MBT
Sequence
1. hAT18486
hAT19037
hAT 2
hAT 3
560 ACCCACCTGGCTCTTGTGTC
AGCGAATGTGTTTTGACCAC
4192 bp TE
hAT 4
Microsatellite (AT)
MBP 81C12 (M. balbisiana) x MA4 82I11 (M. acuminata) BACs.
23/09/2014 12

13. Musa balbisiana (MBP 81C12)
Musa acuminata (MA4 82I11)
hAT 1
1676 TE
384 bp TE + 781 MITE
Transposed Element
Sr. No. Primer Pairs Product Size
(bp)
Microsatellite (AT)
621 bp MBT
Sequence
1. hAT18486
hAT19037
hAT 2
hAT 3
560 ACCCACCTGGCTCTTGTGTC
AGCGAATGTGTTTTGACCAC
4192 bp TE
hAT 4
Microsatellite (AT)
MBP 81C12 (M. balbisiana) x MA4 82I11 (M. acuminata) BACs.
23/09/2014 13

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

15. 23/09/2014 15 Dot plot showing the complete Inverted repeat.

16. 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
23/09/2014 16
1KB
800
600
400
200
hAT1 insertion sites in Musa diversity collection
hAT486F and hAT037R
Top bands (560-bp) amplified hAT element
Lower bands amplifying the flanking sequences only
Menzel, Nouroz, Heslop-Harrison, Schmidt 2014

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

18. IRAP 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.

19. Phylogenetic analysis of Musa genomes – separating species. Teo, Schwarzacher et al.
23/09/2014 19

20. BSV Expression in Banana
Double stranded DNA is infective: Insect vector
Unexpected epidemiology: Appearance after cold or tissue culture

21. Nuclear Copies of Banana Streak Virus in Banana

22. Nuclear Copies
of BSV in banana
DNA Fibre in situ hybridization
Harper, HH et al., Virology 1999 … cf D’Hont et al., Nature, 2012

23. Whole genome shotgun sequencing
• Changing all cytogenomics (.org) work
• Easily obtaining several-fold sequence
coverage

24. D’Hont et al.
Nature 2012
doi:10.1038/nature
11241

25. Musa
Banana
n=11
Sequence:
D’Hont, inc HH et al.
Nature 2012
Haploid: Nair, HH 2013

27. Whole genome duplications
• The surprise to the sequencers: conserved
synteny and relatively few breakpoints
• The surprise to the cytogeneticists:
sequencing shows whole genome duplications
(=polyploidy) deep in the phylogenetic tree
• The surprise to everyone: so few genes but
multifunctional

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

30. Brachiaria
LTR element families
Fabíola Carvalho Santos
André Luiz Laforga Vanzela
See poster
Forage/pasture
Urban
Savanna/cerrado
Forest
Sugar cane
Soybean/corn
Brazil land use

31. Some probes show less
hybridization to some
chromosomes, perhaps indicating
genome specificity.
Fabíola Carvalho Santos
André Luiz Laforga Vanzela
See poster

32. From Chromosome to Nucleus
Pat Heslop-Harrison phh4@le.ac.uk www.molcyt.com

33. Wheat evolution and hybrids
Triticum uratu
2n=2x=14
AA
Aegilops speltoides
Triticum dicoccoides
Einkorn
2n=4x=28
AABB
Triticum monococcum
2n=2x=14
AA
Bread wheat
Triticum
aestivum
2n=6x=42
AABBDD
Durum/Spaghetti
Triticum turgidum ssp durum
2n=4x=28
AABB
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

34. Copyright restrictions may apply.
Inter-retroelement (IRAP)
analysis of Triticum tauschii ssp
tauschii from Iran
SSR/Microsats: all are different
and no tree is supported
Different sequence classes
evolve at different rates
Saeidi, H. et al. Ann Bot 2008 101:855-861; doi:10.1093/aob/mcn042

35. Crop standing
Lodging in cereals
Crop fallen

36. Use of repetitive DNA sequences as
chromosome markers

37. Inheritance of Chromosome 5D
Aegilops ventricosa
DDNN
dpTa1
pSc119.2
Genomic Ae.ventricosa
ABDN
Triticum persicum Ac.1510
AABB
AABBDDNN Marne
AABBDD
VPM1 Dwarf A
CWW1176-4
Rendezvous
Piko
Virtue
96ST61
×
×
×
×
Hobbit
× {Kraka × (Huntsman × Fruhgold)}

38. Wheat Streak Mosaic Virus in North America
Bob Graybosch, USDA

39. Wsm-1: only highly effective source of resistance to WSMV

40. Mace wheat
Graybosch et al. 2009
In situ: Niaz Ali & Schwarzacher

41. Chromosome evolution - Polyploidy
• Selected natural
– Wheat
– Banana
– Brachiaria
– Proso millet
• Synthetic
– Triticale
– Nicotiana

42. Proso millet (Panicum miliaceum):
origins, genomic studies and
prospects
Pat Heslop-Harrison, Farah Badakshi
and Harriet Hunt

43. Panicum sensu stricto c. 100 species; x=9
Evolution of Panicum miliaceum Proso millet
P. virgatum
2n=4x=36 or 2x=18
? ? ? ? ? ?
P. miliaceum
2n=4x=36
P. capillare
2n=2x=18
P. repens
2n=4x=36
also 2n=18 to 54
P. sumatrense
2n=2x=18 or 4x=36
Global North-temperate
Low genetic diverstiy
Weedy forms
• Hunt , HH et al. 2014. Reticulate evolution in Panicum (Poaceae): the
origin of tetraploid broomcorn millet, P. miliaceum. J Exp Bot. 2014

45. • P. miliaceum: allotetraploid with maternal
ancestor P. capillare and one genome shared
with P. repens (also allotetraploid)
 Hunt , HH et al. 2014. Reticulate evolution in Panicum (Poaceae): the origin of
tetraploid broomcorn millet, P. miliaceum. J Exp Bot. March 2014

46. Chromosome
and genome
engineering
Cell fusion
hybrid of two
4x tetraploid
tobacco
species
Patel, Badakshi, HH,
Davey et al 2011 Annals
of Botany

47. Nicotiana
hybrid
4x + 4x
cell fusions
Each of 4
chromosome
sets has
distinctive
repetitive
DNA when
probed with
genomic DNA
Patel et al
Ann Bot 2011
Cell fusion
hybrid of two
4x tetraploid
tobacco
species
Four genomes
differentially
labelled
Patel, Badakshi,
HH, Davey et al
2011 Annals
Botany

48. Arachis hypogaea - Peanut
Tetraploid of recent origin,
ancestors separated only 3 My ago
Ana Claudia Araujo, David Bertioli, TS & PHH EMBRAPA, Brasília. Annals Botany 2013

49. •Arachis hypogea 2n=4x=40 probed with
•(green) A. duranensis; (red) A. ipaënsis
 Bertioli et al. Annals of Botany 2013

50. BAC in situ hybridization

52. Primula BAC mapping
Gilmartin, Lu, HH & Badakshi 2015?

53. 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 (2
radish chromosomes)
far-red 45S rDNA
Rfk1 carrying BAC green
labels sites on radish and
homoeologous pair in
Brassica
Tarja Niemelä,
Seppänen, Badakshi,
Rokka HH
Chromosome Research
2012

54. BACs from different
species have different
repeat distributions –
and hence different
patterns of hybridization

55. Organelle sequences
from chloroplasts or
mitochondria
Sequences from viruses,
Agrobacteriumor other
vectors
Transgenes introduced
with molecular biology
methods
Genes, regulatory and non-coding
single copy sequences
Dispersed repeats:
Transposable Elements
Repetitive DNA sequences
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
Repeated genes
Simple sequence
repeats or
microsatellites
Subtelomeric
repeats
45S and 5S
rRNA genes
Blocks of tandem
repeats at discrete
chromosomal loci
DNA sequence components of the nuclear genome
Heslop-Harrison & Schmidt 2012. Encyclopedia of Life Sciences
Other genes

56. MuTRR
180 bp
X MuTRR
MuTRF
MuTRF
220 bp
Monkey retroelement
• The original 177bp repeat fits nicely around the
nucleosome allowing a tight coiling
• The repeat unit with the retroelement foot print, the
63bp box, has a much more open configuration
• It is maintained as it brings a CG and CNG site that allows
control via methylation
Insertion and
subsequent loss
C.H Teo and Schwarzacher

57. A
B
C
DNA sequence
Centromere
TE
Tandem repeat monomer
TE Transposable element
Single copy DNA
Metaphase
chromosome
Spindle microtubules pulling apart
chromatids
147bp plus 5-70bp linker = 150-220bp
100bp plus 55bp linker = 155bp
D
E
F
G
H
I
Kinetochore
Henikoff et al 2013
C: antibody to CENH3 variant
Heslop-Harrison & Schwarzacher 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

58. Domestication
• Most species domesticated 10,000 years ago:
cereals, legumes/pulses, brassicas, fruits,
cows/sheep/pigs, silkworm/bees)
• Few species more recently (rabbits, fish; trees,
biofuel crops)
• A few dropped out of production
• First steps: productive, reproduce easily,
disease-free, edible/tasty, harvestable …
Heslop-Harrison & Schwarzacher Domestication genomics www.tinyurl.com/domest and
review of rabbits www.tinyurl.com/rabdom

59. Domestication
• …
• A few dropped out of production
• Second steps: more productive, harvestable
• Third step: fitting for sustainable intensification
• Proso millet: the most water-efficient cereal
• Superdomestication and design of crops
Heslop-Harrison & Schwarzacher Domestication genomics www.tinyurl.com/domest and
review of rabbits www.tinyurl.com/rabdom www.tinyurl.com/superdom

60. Outputs
–CROPS
– Fixed energy
Inputs
–Light
–Heat
–Water
–Gasses
–Nutrients
–Light
–Heat
–Water
–Gasses
–Nutrients
(Ecosystem services)

61. Conventional Breeding
• Cross the best with the best and hope for something
better
Superdomestication
• Decide what is wanted and then plan how to get it
– Variety crosses
– Mutations
– Hybrids (sexual or cell-fusion)
– Genepool
– Transformation

62. Economic growth
• Separate into increases in inputs
(resources, labour and capital) and
technical progress
• 90% of the growth in US output per
worker is attributable to technical
progress
Robert Solow – Economist

63. 1.2E+09
1E+09
800000000
600000000
Agronomy
400000000
200000000
0
52 years of plant breeding progress
GM maize
Maize
Rice, paddy
Wheat
Population /10
Genetics
1961 1970 1980 1990 2000 2010 2013

64. United Nations Millennium Development Goals-MDGs
1990 to 2015
• 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

65. From Chromosome to Nucleus
Pat Heslop-Harrison phh4@le.ac.uk www.molcyt.com

66. How do genomes evolve?
– Gene mutation Genome very rarely
evolution
– Chromosome evolution
– Polyploidy and genome duplication (ancient and modern)
– Repetitive sequences: mobility & copy number
– Recombination
– Epigenetic aspects – centromeres & expression
How can we exploit knowledge of genome evolution?
– Biodiversity
– Chromosome and genome engineering
– Breeding
– Markers Pat Heslop-Harrison & Trude Schwarzacher
www.molcyt.com
Pathh1 on slideshare

67. Chromosomes, Crops and
Superdomestication in Katowice
Pat Heslop-Harrison
phh4@le.ac.uk
www.molcyt.com
UserID/PW ‘visitor’
Pathh1:
Twitter #PMC .
Slideshare pathh1

68. Major Genomic Components
• Tandem Repeats
• Simple Sequence Repeats
• Dispersed Repeats
• Functional Repeats
• Retroelements
• Genes
Typical Fraction
10%
5%
10%
15%
50%
10%

70. A D’Hont et al.
Nature 2012
doi:10.1038/na
ture11241
Whole-genome duplication events.

71. Satellite DNA probe green

73. Differences between genomes
Major differences in the nature and amount of
repetitive DNA
• 45S rDNA
• dpTa1 tandem repeat

74. 146 bp around
histones

75. From Chromosome to Nucleus
Pat Heslop-Harrison phh4@le.ac.uk www.molcyt.com

76. • Three copies of the Arabidopsis 180 bp repeat showing (dark purple, stepped line) GC
content of the sequence and (red, smooth line) sequence curvature. While GC and AT
rich regions of a sequence generally correlate with curvature, the kinked region shows
curvature with low GC content.

77. • How do genomes evolve?
• How can we exploit knowledge of genome
evolution?
– Biodiversity
– Chromosome engineering
– Markers

78. Genome engineering
• Introgression of chromosomes
– Brassica – Raphanus
– Wheat – Thinopyrum

79. Chromatin
• Packaging

80. UK Wheat 1948-2007
52,909 data points, 308 varieties
From Ian Mackay, NIAB, UK. 2009. Re-analyses of historical series of variety trials: lessons from
the past and opportunities for the future. SCRI website.

81. Rules for successful domestication
• There aren’t any!
• Crops come from anywhere (new/old world;
temperate/tropical; dry/humid)
• They might be grown worldwide
• Polyploids and diploids (big genomes-small
genomes, many chromosomes-few
chromosomes)
• Seeds, stems, tubers, fruits, leaves

82. DNA methylation is unevenly distributed on
10 m
Musa chromosomes
copia
elements
in methylated
regions, but also
in some low
methylated
regions (arrows)
5MeC

83. DNA methylation is unevenly distributed on
10 m
C.H Teo and Schwarzacher
Musa chromosomes
5MeC
gypsy
elements
in methylated
regions, but also
in some low
methylated
regions (arrows)
Teo &
Schwarzacher in
prep 2013

84. Genome evolution
• How do genomes evolve?
– Mutation very rarely (human: 10−8/site/generation)
– Chromosome evolution
– Polyploidy and genome duplication (ancient and modern)
– Repetitive sequences – mobility & copy number (10−4 μsat)
– Recombination
– Epigenetic aspects – centromeres & expression
• How can we exploit knowledge of genome evolution?
– Biodiversity
– Chromosome engineering
– Breeding
– Markers

85. Outputs
–Crops
(Chemical energy)
– Food
– Feed
– Fuel
– Fibre
– Flowers
– Pharmaceuticals
– Fun 85

86. 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
Molecular cytogenetics …

87. How to use diversity
• Cross two varieties
• Genome manipulations
• Cross two species and make a new one
• Cell fusion hybrids
• Chromosome manipulation
• Backcross a new species
• Generate recombinants
• Chromosome recombinations
• Transgenic approaches
• Use a new species

88. Nothing special about crop 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
Rapeseed B.
napus
1125 Mbp 38 4 Cotyledon oil/protein
Sugar beet 758 Mbp 18 2 Modified root
Cassava 770 Mbp 36 2 Tuber
Soybean 1,100 Mbp 40 4 Seed cotyledon
Oil palm 3,400 Mbp 32 2 Fruit mesocarp
Banana 500 Mbp 33 3 Fruit mesocarp
Heslop-Harrison & Schwarzacher 2012. Genetics and genomics of crop
domestication. In Altman & Hasegawa Plant Biotech & Agriculture. 10.1016/B978-
0-12-381466-1.00001-8
Tinyurl.com/domest

89. DNA sequence
Centromere
TE
Tandem repeat monomer
TE Transposable element
Single copy DNA
Metaphase
chromosome
Spindle microtubules pulling apart
chromatids
147bp plus 5-70bp linker = 150-220bp
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://molcyt.org (Dec 2013)

90. Genes!

91. 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

92. Outputs
–CROPS
– Fixed energy
Inputs
–Light
–Heat
–Water
–Gasses
–Nutrients

93. Outputs
–CROPS
– Fixed energy
93
Inputs
–Light
–Heat
–Water
–Gasses
–Nutrients
– Light
– Heat
– Water
– Gasses
– Nutrients