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Fruit breedomics workshop wp6 molecular markers technology and methods riccardo velasco
- 1. ‘80: DNA manipulation enzymes
- 2. Restriction enzymes ‘80: DNA manipulation enzymes
- 3. Molecular Markers: technology and methods RFLP = Restriction Fragment Length Polymorphisms
- 4. DNA analysis by hybridization DNA ligation and cloning
- 5. ’90: Polymerase Chain Reaction (PCR)
- 8. SSR = Single Sequence Repeats SSLP = Single Sequence Length Polymorphism Molecular Markers: technology and methods
- 9. SNP = Single Nucleotide Polymorphisms Molecular Markers: technology and methods SNP detection
- 10. Methods used to identify and to screen SNPs – 2006-08: low cost-low throughput vs high cost-high throughput Fluorescence-based capillary electrophoresis SSCP capillary electrophoresis P1 P2 Ind1 Ind2 Ind3 Ind4 Ind5 Ind6 I II II III III I III II I II III II II I II Multiplex Minisequencing SNPlex Genotyping System Resequencing P1 P2 Progeny SSCP non-denaturant gelCAPs/dCAPs agarose gel Gel-based electrophoresis P1 P2 Progeny
- 11. IXd02 GCAAGATTCTAATGCCTAAACAGA rev 24mer IXd12 (T)11ACTCCACGAACCCATCCTT rev (19+11) 30mer Va05 (T) 16GGAGGGAAAAGGTGTTCATT for (20+16) 36mer VIc06 (T) 21CTTCTAATGCGCTTTGAATTC for (21+21) 42mer IXd02 CTGGTACGCCTGCAGGTACCGGTCCGGATTCCCGGGTCG ACCCACGCGTCCGCCCACGCGTCCGATCCCAGTTTCCAA GTACACATGTTAAGGAAAAATGGGCAAAGAATCTCTATC TTGCCTGCGTGTGCTTAGGACGAACTGTATCTTGATTAT CCCCTAAACTGCTCAGAAAGGAAGAAACTGTGCC[t/g] CAGACTCTCCCTTCTTTCCATTGTCTGTTTCCTTCATGT TAATTGTAAAAATTGGTGGGTGAGAGCT[a/g]TCTGTT TAGGCATTAGAATCTTGCTCCCATGTATGATAAACTATT TTTATGACAGCGTTTTTTTTGTTGTTGGCCCTTACTCTC CAGCCTTAGATGCTGCCATTTGTAGAAGAAGGCTTTTGT TTTTATAATTGCAATTTAAAATTGATGGATAGTCGGATG Va05 CGCCTGCAGGTACCGGTCCGGATTCCCGGGTCGACCCACG CGTCCGAAGGGAGCAAGAGGTGCTTCTGGCCTTTGTATTG CCCATGGAGGTGGTCGGAGGTGCCAGAAAACTGGATGTCA TAAGGGAGCTGAGGGCCGTACAGTGTACTGCAAGGCACAT GGGGGTGGCCGGCGATGTGAATTCCTTGGGTGCACAAAGA GTGCAGAAGGACGAACAGATTACTGCATTGCTCATGGTGG TGGCAGGCGATGCAGTCACGAGGGTTGCACTCGAGCTGCA AGAGGGAAATCAGGATTGTGCATCCGGCATGGTGGGGGGA AGAGATGCAAGAAGGAGAACTGCACGAAGAGTGCAGAAGG CCTCTCAGGTCTCTGCATCTCACACGGAGGTGGGCGTCGT TGCCAGTTCCCAGCATGCACAAAAGGGGCACAAGGAAGCA CAATGTATTGCAAGGCCCATGGTGGGGGAAAACGGTGCAC AGTTCCAGG[g/t]TGCACCAAGGGCGCAGAAGGGAGCAC CCCCTTCTGCAAGGGGCATGGTGGAGGGAAAAGGTGTTCA TT[c/t]CAAGGTGGTGGGATTTGCCCAAAGAGTGTTCAT GGAGGGACCAACTTCTGCGTGGCACATGGGGGCGGCAAGA GGTGTGCAGTGCCAGAATGCACAAAGAGCGCAAGGGGACT GCAAGATTCTAATGCCTAAACAGA …...GCTATTCGTTCTAAGATTACGGATTTGTCTAGTTTAGG….... T C A G dd dd dd dd Minisequencing
- 12. The final product is a fragment elongated by 1 single nucleotide (~18 to 26 bases) T/T homozygote G/T heterozygote G/G homozygote Minisequencing
- 13. Syrah Pinot noir 87 229 216 227 127 106 Fond. Mach di San Michele all’Adige – Sequencing platform High throughput methods (2007)
- 14. Syrah Pinot 22 30 36 42 48 54 G/G C/C C/T G/T A/T G/A G/A C/T C/T G/G A/A G/G SNPs multiplexing
- 15. Marker type Cost per data point Cost per sample Main use n. of individuals/ output Pro(*) Contra(*) RFLP low low mapping low co-dominant AFLP very low low mapping medium dominant SSR medium medium mapping/ genotyping high co-dominant SCAR medium medium genotyping high co-dominant SNP: reSeq high medium mapping high co-dominant Kasp low low genotyping high co-dominant CHIP very low high mapping/ genotyping extremely high co-dominant dominant Small array low medium mapping/ genotyping very high co-dominant dominant (*) dominant: yes/no; co-dominant: aa; ab; bb
- 17. SNPlex Genotyping System (Applied Biosystems) ABI SNPlexTM OLA Genotyping Annealing/Ligation 5’ & 3’ Exonuclease PCR/Biotin Labeling Coated Microtiter Plate Labeled & Allele-specific A set of universal core reagent kits and a set of SNP-specific ligation probes - Two allele-specific oligos ASO (two for each SNP) - One locus-specific oligo LSO 48-plex= 144 probes
- 18. High-throughput PCR-based method 64 x 384 well plates = 24.576 BACs Six different matrices 32 PP, 32 FP, 24 SP 32 RP, 32 CP, 32 DP 184 POOLS = 24.576 BACs 24.576 BACs in 2 x 96 well plates Screened with ESTs, SSRs, AFLPs Klein et al., Gen. Res. (2000)Screening efficiency 87% SSR, 84% ESTs
- 19. Screening of SSRs, SNPs, AFLP markers Analyzed using Bionumerics software BAC ends sequencing SSRs & SNPs AFLP (Keygene) + Continue……….