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Chromosomes and molecular cytogenetics of oil palm: impact for breeding and genetics PIPOC

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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.

Published in: Science
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Chromosomes and molecular cytogenetics of oil palm: impact for breeding and genetics PIPOC

  1. 1. Oil palm molecular cytogenetics & genomics KLCC 7th October 2015 Pat Heslop-Harrison phh@molcyt.com www.molcyt.com Twitter, YouTube and Slideshare
  2. 2. 10/6/2015 Oil Palm 32 chromosomes DAPI; TTTAGGG telomere; 45S rDNA (1 major pair + minor) 5S rDNA (1 major + minor)
  3. 3. Telomere: (TTTAGGG)n Centromere: function Genes: small proportion
  4. 4. The Karyotype and Repetitive DNA Several repeats are broadly proximal, others cover whole arms
  5. 5. The Karyotype and repetitive DNA SSRs tend to be distal in oil palm
  6. 6. 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
  7. 7. 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!
  8. 8. 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
  9. 9. 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
  10. 10. Green: Tat transposable element in polyploid Brachiaria tropical forage grass Fabiola Santos et al. 2015 Chromosome Research 15
  11. 11. Green: Transposable element locations in diploid Brachiaria tropical forage grass Fabiola Santos et al. 2015 Chromosome Research 16
  12. 12. 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
  13. 13. 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.
  14. 14. Major Genomic Components Tandem Repeats Simple Sequence Repeats Dispersed Repeats Functional Repeats Retroelements Genes Typical Fraction 10% 5% 10% 15% 50% 10%
  15. 15. Epigenetics Phenotype appears 5 years after tissue culture
  16. 16. 06/10/2015 Modulation of Methylation  McrBC - shows substantial reduction in methylation in tissue culture lines  Cuts methylated DNA
  17. 17. 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)
  18. 18. 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
  19. 19. Modulation of Methylation  McrBC - shows substantial reduction in methylation in tissue culture lines  Cuts methylated DNA
  20. 20. 10/6/2015 Modulation of Methylation  McrBC digests probed with gypsy clones  present only in N and T lines  Similar with copia probe
  21. 21. 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
  22. 22. 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
  23. 23. 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)
  24. 24. Search for Methyltransferase Homologues Probed with Arabidopsis clones
  25. 25. 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
  26. 26. 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
  27. 27. 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
  28. 28. 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
  29. 29. 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
  30. 30. Oil palm molecular cytogenetics & genomics KLCC 7th October 2015 Pat Heslop-Harrison phh@molcyt.com www.molcyt.com Twitter, YouTube and Slideshare
  31. 31. 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.
  32. 32. 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
  33. 33. From Chromosome to Nucleus Pat Heslop-Harrison phh4@le.ac.uk www.molcyt.com
  34. 34. Find and use biodiversity 39
  35. 35. Outputs –CROPS – Fixed energy 40 Inputs –Light –Heat –Water –Gasses –Nutrients – Light – Heat – Water – Gasses – Nutrients

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