1. Use of SNP-DNA analysis in authenticating Basmati rice
From waaay back in …with a font that was fashionable then

2. Single nucleotide polymorphisms
Single nucleotide differences exist between individuals, plant and animal varieties and bacterial strains
TCGAAATGCATGCGCATGATT
Variety 1
TCGAAATGCATGCGCGTGATT
Variety 2

3. Assaying SNPs by MALDI-ToF MS
Once a SNP is identified, 3 primers are required to enable genotyping including:-
- Two PCR primers to amplify the region around the SNP
5’ ’
- One Extension primer which anneals directly adjacent to the SNP.
5’ ’

4. Assaying SNPs by MALDI-ToF MS
TCGAAATGCATGCGCATGATT
TCGAAATGCATGCGCGTGATT
Anneal primer
TCGAAATGCATGCGCATGATT
TCGAAATGCATGCGCGTGATT
TTACGTACGCG
+polymerase
+ddNTP’s
TCGAAATGCATGCGCATGATT
TCGAAATGCATGCGCGTGATT
TTACGTACGCGT
TTACGTACGCGC
Analyse extension products by MALDI-TOF MS

5. MALDI-TOF Mass Spectrometry
Electric potential across the flight tube to the detector.
Larger ions take longer to fly.
This Time of Flight (ToF) allows the separation of ions on the basis of size.
Samples spotted onto a MALDI plate
Nitrogen laser (337nm) vaporises and ionises the sample.

6. Difference in peak size reveals the SNP allele for that rice variety
Assaying SNPs by MALDI-ToF MS
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

7. Multiplexing SNP assays

8. Quantitative SNP assays

9. MALDI-TOF-based DNA authentication of basmati rice
Sponsored by the FSA

10. FSA sponsored work includes:-
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

11. Basmati rice varieties
Traditional breeds
Basmati 386
Basmati 370
Dehradun
Ranbir Basmati
Taraori (HBC-19)
Hybrid
Basmati 385
Super Basmati
Pusa Basmati
Kasturi
Basmati 198
Common Adulterants
Pusa 169
Sabarmati
PR 106
Kali-much
Lakra
Saket 4
Parimal
Sherbati
Terricot

12. Assembling a SNP database for Basmati varieties and adulterants

13. Examples of survey work

14. SNP Genotyping using primer I
Primer + C
Primer + T
Tilda basmati
Rice
ASDA basmati
rice

15. SNP Genotyping using 11 primers
Tilda basmati rice behaves like traditional variety Kernal
Asda basmati rice suggests another hybrid: Pusa

16. SNP analysis as a QC tool
Plausible contamination / mixture (S + T = 2003) ?

17. from mixing (or crossing)
SNP analysis as a QC tool
New SNP marker alleles
Revealed that the
2003 crop
could NOT have arisen
from mixing (or crossing)
Super Basmati (S)
with Thai (T) rice

18. HumanOmniExpress-24 BeadChip
Number of Markers >713,014
HumanOmniExpress-24 BeadChip
Zoom ahead to 2012
Manhattan plot of association with cilantro soapy-taste from 14,604 unrelated participants