fruit breedomics workshop wp6 molecular markers technology and methods riccardo velasco
TRANSCRIPT
‘80: DNA manipulation enzymes
Restriction enzymes
‘80: DNA manipulation enzymes
Molecular Markers: technology and methods
RFLP = Restriction Fragment Length Polymorphisms
DNA analysis by hybridization
DNA ligation and cloning
’90: Polymerase Chain Reaction (PCR)
SSR = Single Sequence RepeatsSSLP = Single Sequence Length Polymorphism
Molecular Markers: technology and methods
SNP = Single Nucleotide Polymorphisms
Molecular Markers: technology and methods
SNP detection
Methods used to identify and to screen SNPs – 2006-08: low cost-low throughput vs high cost-high throughput
Fluorescence-based capillary electrophoresisSSCP capillary electrophoresis
P1
P2
Ind1
Ind2
Ind3
Ind4
Ind5
Ind6
I II
IIIII
IIII
III II
I II
IIIII
II
I II
Multiplex Minisequencing
SNPlex Genotyping System
Resequencing
P1 P2Progeny
SSCP non-denaturant gelCAPs/dCAPs agarose gel
Gel-based electrophoresis
P1 P2 Progeny
IXd02GCAAGATTCTAATGCCTAAACAGA rev 24mer
IXd12(T)11ACTCCACGAACCCATCCTT rev (19+11) 30mer
Va05(T) 16GGAGGGAAAAGGTGTTCATT for (20+16) 36mer
VIc06(T) 21CTTCTAATGCGCTTTGAATTC for (21+21) 42mer
IXd02CTGGTACGCCTGCAGGTACCGGTCCGGATTCCCGGGTCGACCCACGCGTCCGCCCACGCGTCCGATCCCAGTTTCCAAGTACACATGTTAAGGAAAAATGGGCAAAGAATCTCTATCTTGCCTGCGTGTGCTTAGGACGAACTGTATCTTGATTATCCCCTAAACTGCTCAGAAAGGAAGAAACTGTGCC[t/g]CAGACTCTCCCTTCTTTCCATTGTCTGTTTCCTTCATGTTAATTGTAAAAATTGGTGGGTGAGAGCT[a/g]TCTGTTTAGGCATTAGAATCTTGCTCCCATGTATGATAAACTATTTTTATGACAGCGTTTTTTTTGTTGTTGGCCCTTACTCTCCAGCCTTAGATGCTGCCATTTGTAGAAGAAGGCTTTTGTTTTTATAATTGCAATTTAAAATTGATGGATAGTCGGATG
Va05CGCCTGCAGGTACCGGTCCGGATTCCCGGGTCGACCCACGCGTCCGAAGGGAGCAAGAGGTGCTTCTGGCCTTTGTATTGCCCATGGAGGTGGTCGGAGGTGCCAGAAAACTGGATGTCATAAGGGAGCTGAGGGCCGTACAGTGTACTGCAAGGCACATGGGGGTGGCCGGCGATGTGAATTCCTTGGGTGCACAAAGAGTGCAGAAGGACGAACAGATTACTGCATTGCTCATGGTGGTGGCAGGCGATGCAGTCACGAGGGTTGCACTCGAGCTGCAAGAGGGAAATCAGGATTGTGCATCCGGCATGGTGGGGGGAAGAGATGCAAGAAGGAGAACTGCACGAAGAGTGCAGAAGGCCTCTCAGGTCTCTGCATCTCACACGGAGGTGGGCGTCGTTGCCAGTTCCCAGCATGCACAAAAGGGGCACAAGGAAGCACAATGTATTGCAAGGCCCATGGTGGGGGAAAACGGTGCACAGTTCCAGG[g/t]TGCACCAAGGGCGCAGAAGGGAGCACCCCCTTCTGCAAGGGGCATGGTGGAGGGAAAAGGTGTTCATT[c/t]CAAGGTGGTGGGATTTGCCCAAAGAGTGTTCATGGAGGGACCAACTTCTGCGTGGCACATGGGGGCGGCAAGAGGTGTGCAGTGCCAGAATGCACAAAGAGCGCAAGGGGACT
GCAAGATTCTAATGCCTAAACAGA…...GCTATTCGTTCTAAGATTACGGATTTGTCTAGTTTAGG…....
T
C A
G
dd
dd
dd
dd
Minisequencing
The final product is a fragment elongated by 1 single nucleotide (~18 to 26 bases)
T/Thomozygote
G/Theterozygote
G/Ghomozygote
Minisequencing
Syrah Pinot noir
87 229 216
227 127 106
Fond. Mach di San Michele all’Adige – Sequencing platform
High throughput methods (2007)
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
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
PCR-based KASP™ genotyping assay
SNPlex Genotyping System (Applied Biosystems)
ABI SNPlexTM OLA Genotyping
Annealing/ Ligation
5’ & 3’ Exonuclease
PCR/ Biotin Labeling
Coated Microtiter Plate
Labeled & Allele-specifi c
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
High-throughput PCR-based method
64 x 384 well plates = 24.576 BACs
Six different matrices32 PP, 32 FP, 24 SP32 RP, 32 CP, 32 DP
184 POOLS = 24.576 BACs
24.576 BACs in 2 x 96 well plates
Screened withESTs, SSRs, AFLPs
Klein et al., Gen. Res. (2000)Screening efficiency 87% SSR, 84% ESTs
Screening of SSRs, SNPs, AFLP markers
Analyzed using Bionumerics software
BAC ends sequencing
SSRs & SNPs
AFLP (Keygene)
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Continue……….
Data Integration
GR05680,0
GR01767,2
BA002517,6BA000321,1F20236b21,8IN012623,4GR040924,4GR028025,5F2068126,1E39/M49-11426,7E32/M62-28230,5F20236a33,7
Chr 10