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In situ hybridization results and examples for course Trude Schwarzacher

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

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In situ hybridization results and examples for course Trude Schwarzacher

  1. 1. Trude Schwarzacher Department of Biology University of Leicester, UK ts32@le.ac.uk www.molcyt.com
  2. 2. Background and history  What is it?  What can it do?  Applications  Examples from our lab  Wheat  Brassica  Crocus
  3. 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. 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
  5. 5. 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.
  6. 6.  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
  7. 7. 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
  8. 8. www.CrocusBank.org
  9. 9. Saffron Crocus sativus 2n=3x=24 C. cartwrightianus green C. thomasii red
  10. 10. Triticeae (Hordeae) Small grain cereals in the family of Poaceae (grasses) wheat barley rye Einkorn Spelt wheat Leymus
  11. 11. Triticeae phylogeny
  12. 12. 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
  13. 13. 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
  14. 14. 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
  15. 15. 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
  16. 16. 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
  17. 17. NOR: rDNA loci vary in number, position and size Chromosomal Satellite
  18. 18. 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
  19. 19. 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
  20. 20. 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
  21. 21. 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
  22. 22. 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.
  23. 23. 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
  24. 24. Rye DNA pTa71-45S rDNA 4 major sites 1RS, 6BS 6 major sites 1RS, 1BS, 6BS 1BL.1RS 1DL.1RS
  25. 25. rye chromosome derivative 1R substitutes wheat chromosome 1D DAPI Rye genomic DNA probe pTa71 (45s rDNA probe) Forsström and Schwarzacher 2000
  26. 26. Derivative chromosome 1R of Lines 7-102 and 7-169
  27. 27. The genusThynopyrum, including wild goat grasses and wheat grasses, has proven an excellent source for disease and biotic stress resistance
  28. 28. 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
  29. 29. 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
  30. 30. O. violaceus genomic DNA DAPI Xianghong Ge, Farah Badakshi, Heslop-Harrison and Schwarzacher 2010
  31. 31.  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
  32. 32. Triticeae genomes and chromosomes 2n = 14 Secale cereale , rye 7 chromosome pairs R genome 1 2 3 4 5 6 7
  33. 33. 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)
  34. 34. 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
  35. 35. Telomere (TTTAGGG)n Universal in eukaryotes with only a few exceptions Dynamic Number of repeats varies: tissue, age and chromosome Added by telomerase
  36. 36. Rye, subteloemreic sequences
  37. 37. Differences between genomes Major differences in the nature and amount of repetitive DNA • pTa71 • dpTa1
  38. 38. 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
  39. 39. 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
  40. 40. 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)
  41. 41. 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
  42. 42. Tandem Repeats
  43. 43. 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
  44. 44. 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

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