Use of SNP-DNA analysis in authenticating Basmati

rice

• DNA based authentication of basmati rice • Background to Single nucleotide

polymorphisms (SNPs)• Introduction to SNP genotyping using

MALDI-ToF MS • Authentication of basmati rice using

MALDI-ToF MS-based SNP genotyping• Launching a commercial genotyping service

Summary

Authentication of basmati rice

by DNA genotyping • Micro-satellite markers

• Single nucleotide polymorphisms

TCGAAATGCATGCGCATGATTVariety 1

TCGAAATGCATGCGCGTGATTVariety 2

Single nucleotide differences exist betweenindividuals, plant and animal varieties

and bacterial strains

Single nucleotide polymorphisms

Authentication using SNPs

• PCR based SNP genotyping

• MALDI-ToF MS based SNP genotyping

Single nucleotide polymorphisms

Once a SNP is identified, 3 primers are required to enable genotyping including:-

- Two PCR primers to amplify the region around the SNP

5’ 3’

- One Extension primer which anneals directly adjacent to the SNP.

5’ 3’

Assaying SNPs by MALDI-ToF MS

TCGAAATGCATGCGCATGATT TCGAAATGCATGCGCGTGATT

TCGAAATGCATGCGCATGATT TCGAAATGCATGCGCGTGATTTTACGTACGCG TTACGTACGCG

TCGAAATGCATGCGCATGATT TCGAAATGCATGCGCGTGATTTTACGTACGCGT TTACGTACGCGC

+polymerase+ddNTP’s

Anneal primer

Analyse extension products by MALDI-TOF MS

Assaying SNPs by MALDI-ToF MS

MALDI-TOF Mass Spectrometry

Samples mixed with a matrix in great excess and spotted onto a MALDI plate and loaded into the Mass Spectrometer.

Sample spot is subjected to repeated pulses of a nitrogen laser at 337nm in a vacuum which vaporises and ionises sample. Matrix absorbs the majority of incident laser energy, preventing degradation or fragmentation of the sample, and allows the vaporisation and ionisation of some of the substrate.

Delayed application of an electric field causes ions to enter flight tube and accelerate towards detector. Delayed Extraction applies high voltage electric pulse after predetermined time delay to minimise energy spread of ions. All ions gain same kinetic energy, so larger ions take longer to reach detector. This variation in Time of Flight allows the separation of ions on the basis of size.

Assaying SNPs by MALDI-ToF MS

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C = 273 Da

T = 288 Da

A = 297 Da

G = 313 Da

Difference in peak size reveals the SNP

allele for that rice variety

Unextended SNP primer

Extended SNP primer

Multiplexing SNP assays

Quantitative SNP assays

MALDI-TOF-based DNA authentication of basmati

rice

Sponsored by the FSA

• Completing a SNP database for the main commercial varieties and their adulterants.

• Assessing the reproducibility of the protocols to determine the uncertainty of the method.

• Performing a limited survey of Basmati Products being sold in UK supermarkets.

• Developing a Standard Operating Procedure

FSA sponsored work includes:-

Basmati rice varieties

Traditional breeds

Basmati 386Basmati 370DehradunRanbir BasmatiTaraori (HBC-19)

HybridBasmati 385Super BasmatiPusa BasmatiKasturiBasmati 198

Common AdulterantsPusa 169SabarmatiPR 106Kali-muchLakraSaket 4ParimalSherbati

Terricot

RICE VARIETY Basmati 370 Derhadun Kernal PK370 l Pak 385 Pusa 1 Sherbati Super TaraoriLINEAGE Pure Bred Pure Bred Pure Bred Pure Bred Hybrid Hybrid Non-Bas Hybrid Pure Bred

ory-122-1 GA GG AA GG GG AA GG GG GA

ory-58-2 AA AA AA AA TT AA TT AA AA

ory-262 GG GG GG GG GG GG CC GG GG

ory-S0186 TT TT TT TT TT CC CC TT TT

ory-S0157 GG GG GG GG GG CG CC GG GG

ory-S0153 GG GG GG GG GG GG CC GG GG

ory-462 GA GA GA Ga GA GA GA GA GAory-592 GG GG AA GG GG AA GG GG AA

ory-599 TC TC TC TC CC CC CC TC TCory-624-1 GG GG GG GG CC GG GC GG GG

ory-668 AG AA AA GA GG GG GG AA AA

Assembling a SNP database for Basmati varieties and adulterants

Assessing reproducibility

Examples of survey work

SNP Genotyping using primer I

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Primer Primer + TPrimer + C

Tilda basmatiRice

ASDA basmatirice

SNP Genotyping using 11 primers

Tilda D6 Asda D5 Kernal (a1)A AA AA AAB AA AA AAC GG GG GGD TT CC TTE GG CC GGF GG GG GGG GA GA GAH AA AA AAI TC CC TCJ GG GG GGK AA GG AA

Tilda basmati rice behaves like traditional variety Kernal

Asda basmati rice is most like the basmati hybrid Pusa

MALDI-ToF MS based genotyping represents an effective analytical

approach to assess:-

• Product authentication using a diagnostic panel of multiplexed SNP markers based on an expanded database.

• Product adulteration, exploiting the techniques ability to quantitate SNPs in the 5-100% range.

Summary

Marker Super Thai 2003ory-122-1 GG GG GGory-58-2 AA AA ATory-262 GG GG CGory-S0186 TT TT CTory-S0157 GG GG CGory-S0153 GG CC GGory-462 AG AG AGory-592 GG AA GGory-599 CT CT CC ory-624-1 GG GG CGory-668 AA AA AG

SNP analysis as a QC tool

S+T 2003GG GGAA ATGG CGTT CTGG CGGC GGAG AGGA GGCT CC GG CGAA AG

SNP analysis as a QC tool

SNP markers revealed that results with the 2003 crop

could NOT have arisen from mixing

Super Basmati (S) with Thai (T) rice

Applying cutting-edge DNA technology to provide analytical services

for product authentication to the food industry.

http://Authentigene.comhttp://Authentigene.info

THE gold standard

Providing a commercial genotyping service

The Authentigene team

Prof. David Archer Prof. Malcolm Bennett Prof. Ian ConnertonDr. Sean May Prof. Gregory Tucker