See also related talk Crops, Climate Change and Super-domestication Heslop-Harrison for Oil Palm Breeders symposium on Gearing Oil Palm Breeding and Agronomy for Climate Change: Keynote opening address MPOB PIPOC and PIPOC ISOPB ISOPA http://www.slideshare.net/PatHeslopHarrison/heslop-harrisoncrops-climatechangesuperdomestication Molecular cytogenetic analysis of the chromosomes of oil palm allows us to understand their evolution, genetics and segregation, genetic recombination and karyotypic stability. The cytogenetic manipulation of genomes and their chromosomes is often valuable for plant breeders to introduce and exploit new variation. Cytological landmarks such as centromeres, telomeres, heterochromatin and nucleolar organizer regions are important for the integration of physical chromosomes with the DNA sequence information. This linkage of the genetic, chromosomal and physical maps is particularly useful in a long-lived tree crop where genetic mapping requires decades of preparation and the mapping crosses may not be directly relevant to DxP commercial plantings. Repetitive DNA is often the most rapidly evolving genomic component, but is poorly understood from sequence assemblies; molecular cytogenetic studies allow its organization and variation to be studied, and the exploitation of repetitive sequences as markers and, by the amplification and mobility of transposable elements or satellite repeats, in generation of new variation. Molecular cytogenetic approaches provide tools for oil palm genomic research, comparative genomics and evolutionary studies and further facilitate understanding the inheritance of specific traits in oil palm, including DNA methylation, epigenetics, and somaclonal variation, allowing work with hybrids, haploids and polyploids. Knowledge of the structures and organization of the chromosomes of oil palm, as in many crop species, is valuable for development of new lines, making hybrids, understanding the causes of some abnormalities or infertility, and exploiting variation and biodiversity found in related species or breeding lines. Further information and slides from the talk will be on our website www.molcyt.com.

1. Oil palm molecular
cytogenetics & genomics
KLCC 7th October 2015
Pat Heslop-Harrison
phh@molcyt.com
www.molcyt.com
Twitter, YouTube and Slideshare

2. 10/6/2015
Oil Palm
32 chromosomes DAPI;
TTTAGGG telomere;
45S rDNA (1 major pair + minor)
5S rDNA (1 major + minor)

3. Telomere: (TTTAGGG)n
Centromere: function
Genes: small proportion

4. The Karyotype and
Repetitive DNA
Several repeats are broadly proximal,
others cover whole arms

6. The Karyotype and repetitive DNA
SSRs tend to be distal in oil palm

11. Organellar
Repetitive DNA
sequences
Plant Nuclear
Genome
DNARetro-
Telomeres
Microsatellites
DNA sequence components of the plant nuclear genome
Heslop-Harrison & Schmidt 2012. Encyclopedia of Life Sciences
Organellar
Viral
Transgenes
Genes
Dispersed:
Transposable
Elements
Tandem
Centro-
meres
Structural
Telo-
meres
Micro-
satellites
Repeated
genes
Sub-
telomeric
rRNA
Blocks
Others

12. Organelle sequences
from chloroplasts or
mitochondria
Sequences from viruses,
Agrobacterium or other
vectors
Transgenes introduced
with molecular biology
methods
Genes, regulatory and non-
coding single copy sequences
Dispersed repeats:
Transposable Elements
Repetitive DNA sequences
Plant 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
Simple sequence
repeats or
microsatellites
Repeated genes
Subtelomeric
repeats
45S and 5S
rRNA genes
Blocks of tandem
repeats at discrete
chromosomal loci
DNA sequence components of the plant nuclear genome
Heslop-Harrison & Schmidt 2012. Encyclopedia of Life Sciences
http://molcyt.org/2012/08/18/plant-nuclear-genome-composition/
Other genes
Plant genome components – full for Slideshare!

13. Repetitive DNA:
The major genomic component
Actively evolving, amplifying and moving
Major consequences for gene expression and
genome behaviour
Modern sequencing:
Current methods 'mask' out repeats and collapse
or jump across so not
Included in assembly
Molecular cytogenetic approaches and new
analyses let us understand the organization,
variation and consequences of these sequences
Repeats in assemblies cause problems

14. Major domains from 9 diverse retroelement families
Chromosomal-distribution-and-evolution-of-retrotransposons-in
-diploid-and-polyploid-Brachiaria-forage-grasses
Fabiola Santos et al. 2015 Chromosome Research

15. Green: Tat transposable element in polyploid Brachiaria tropical forage grass
Fabiola Santos et al. 2015 Chromosome Research 15

16. Green: Transposable element locations in diploid
Brachiaria tropical forage grass
Fabiola Santos et al. 2015 Chromosome Research 16

17. Solanaceae – potato, tomato, peppers, petunia
Petunia Leader Cris Kuhlemeier with Quattrocchio, Sims, Mueller et al.
Repetitive DNA analysis
Katja Richert-Pöggeler, Trude Schwarzacher, Pat Heslop-Harrison
P. inflata P. hybrida P.axillaris

18. Please do not photograph
Petunia data confidential
Reference sequences:
Hansen & Heslop-Harrison Adv Bot Res 2004
http://www.le.ac.uk/bl/ts32/pubs/hhretros.pdf
Petunia genome paper: Kuhlemeier et al.

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

20. Epigenetics
Phenotype appears 5 years after tissue culture

21. 06/10/2015
Modulation of
Methylation
 McrBC - shows
substantial reduction in
methylation in tissue
culture lines
 Cuts methylated DNA

22. January 23, 1999 - PORIM -Molecular Cytogenetics - 22
Modulation of
Methylation
Preliminary results with
anti-methyl-cytosine
indicate differences
between ortet right,
more methylated) &
mantled regenerants
(left, less methylated)

23. January 23, 1999 - PORIM -Molecular Cytogenetics - 23
Modulation of Methylation: Status
Changes seen in retroelements in culture
some reverting in regenerants
New DNA methylation pattern
Expression of copia

24. Modulation of
Methylation
 McrBC - shows
substantial reduction in
methylation in tissue
culture lines
 Cuts methylated DNA

25. 10/6/2015
Modulation of
Methylation
 McrBC digests probed
with gypsy clones
 present only in N and
T lines
 Similar with copia
probe

26. Variation of LINEs between varieties
1. Species1
2-12.Species2
13.Musa
HindIII digests
M:/EcoRI+HindIII
Similar for
gypsy-elements
and En/Spm
transposons
Yes between varieties, Maybe between parent and regenerants

27. Gypsy-element pEgKB7
Ortet | Regenerant
 Marginal
differences
seen in HpaII
(cuts CCGG)
and MspI
(cuts Cm
CGG too)
digests
 Consistent with
HPLC data
 Tracks:
HindIII HaeIII ApaI RsaI HpaII MspI

28. January 23, 1999 - PORIM -Molecular Cytogenetics - 28
Modulation of Methylation after tissue culture
anti-methyl-cytosine : ortet right, more methylated)
mantled regenerants (left, less methylated)

29. Search for Methyltransferase Homologues
Probed
with
Arabidopsis
clones

30. The Molecular Cytogenetics of Somaclonal
Variation in Oil Palm:
Karyotypes / Repetitive DNA
(Retro)transponsons and Dispersion
copia, gypsy, LINE, En/Spm
various characteristic copy numbers and variation
copia and LINEs show ‘activation’ in culture
Methylation / Modulation

31. The Molecular Cytogenetics of Somaclonal
Variation in Oil Palm:
Karyotypes / Repetitive DNA
Retrotransponsons and Dispersion
Methylation / Modulation
CCGG, HPLC, McrBC, antibodies
Retroelement differences and expression

32. Application of molecular cytogenetics
that to oil palm
‘Assessing the presence’: DNA sequence,
chromosomal distribution, diversity
‘Beyond the sequence’: The contribution of
methylation, chromatin packing,
recombination and retroelements to
genome behaviour
‘Genome archaeology’: What has happened
during evolution and what changes now

33. 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

34. There is more information in the
genome than in the sequence alone
‘Epigenetics’
DNA modification
Histone modification
Chromatin packaging
Nuclear architecture
The physical position of a
gene matters

35. Oil palm molecular
cytogenetics & genomics
KLCC 7th October 2015
Pat Heslop-Harrison
phh@molcyt.com
www.molcyt.com
Twitter, YouTube and Slideshare

36. Retroelements, transposons and methylation
status in the genome of oil palm (Elaeis guineensis)
and the relationship to somaclonal variation.
36
 Plant Mol Biol. 2003 May;52(1):69-79.
 Kubis SE1, Castilho AM, Vershinin AV, Heslop-Harrison JS.
 We isolated and characterized different classes of transposable DNA elements in oil palm (Elaeis guineensis) plants grown from seed, and
plants regenerated from tissue culture that show mantling, an abnormality leading to flower abortion. Using PCR assays, reverse
transcriptase fragments belonging to LINE-like and gypsy-like retroelements and transposase fragments of En/Spm transposons were cloned.
Sequence analysis revealed the presence of a major family of LINEs in oil palm, with other diverged copies. Gypsy-like retrotransposons form
a single homologous group, whereas En/Spm transposons are present in several diverged families. Southern analysis revealed their presence
in low (LINEs) to medium (gypsy and En/Spm) copy numbers in oil palm, and in situ hybridization showed a limited number of distinct loci for
each class of transposable element. No differences in the genomic organization of the different classes of transposable DNA elements
between ortet palm (parent) and regenerated palm trees with mantled phenotype were detected, but different levels of sequence
methylation were observed. During tissue culture, McrBC digestion revealed the genome-wide reduction in DNA methylation, which was
restored to near-normal levels in regenerated trees. HPLC analysis showed that methylation levels were slightly lower in the regenerated
trees compared to the ortet parent. The genomic organization of the transposable DNA elements in different oil palm species, accessions
and individual regenerated trees was investigated revealing only minor differences. The results suggest that the mantled phenotype is not
caused by major rearrangements of transposable elements but may relate to changes in the methylation pattern of other genomic
components.

37. Repetitive DNA and the Chromosomes in
the Genome of Oil Palm (Elaeis guineensis)
 A. CAST ILHO, A. VERSH IN IN and J. S. HESLOP -HARRISON
 Annals of Botany 85: 837-844, 2000 doi:10.1006/anbo.2000.1145
Like most plant genomes, much of the oil palm genome (Elaeis guineensis L., 2n ˆ32) consists of repetitive DNA
sequences. We aimed to isolate and characterize a range of repetitive sequences from the genome of the crop and
analyse the repeats by sequencing, Southern and in situ hybridization. Three unrelated repetitive sequence
families, with no homology to known sequences, showed a dispersed distribution along the chromosomes with
concentration in the proximal parts of arms, while simple sequence repeats of DNA (GA, GATA and CAC) were
clustered in the distal parts. Copia-like retroelements were dispersed throughout the genome, with a concentration
in proximal regions, but were not as abundant as in species with larger genomes. Among tandemly repeated
sequences, a major 18S-25S rDNA site was present on a single pair of chromosomal sites, often on a satellite with
no visible connection to its parent chromosome. A major 5S rDNA site was located on another chromosome pair;
variable numbers of minor sites of both rDNA families were also detected. The telomeric sequence (CCCTAAA) was
located at the ends of all chromosome arms, but no intercalary sites of ampli®cation were detected. No other major
families of tandemly repeated sequences were found. The molecular cytogenetic analysis and chromosome
ampli®cation patterns of major sequence families provide the reference point for examination of genomic
organization of major classes of the repetitive DNA in normal and in tissue culture material including abnormal
regenerants. Annals of Botany
Key words: Oil palm, genome evolution, chromosome evolution, repetitive DNA, retrotransposons, genetic markers
37

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

39. Find and use biodiversity 39

40. Outputs
–CROPS
– Fixed energy
40
Inputs
–Light
–Heat
–Water
–Gasses
–Nutrients
– Light
– Heat
– Water
– Gasses
– Nutrients