‘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