SNP Genotyping Assays and Platfoms

Nagendra Singh

ICAR-NRCPB New Delhi

NBPGR ICAR-HRM Training Lecture 14 March 2018

Outline

1. What is SNP?

2. Molecular origin of SNP

3. Methods of SNP Discovery

4. SNP Genotyping Assays and

Platforms

5. SNP Applications

1.What is SNP?

What is a SNP? •Single nucleotide polymorphism (SNP)

•A single base position in a chromosomal locus at which the DNA sequence can vary in populations- e.g. some individuals have “C” and others have “G” at a particular position

•SNPs are normally bi-allelic , i.e. there is a choice of just two bases at the SNP position

•Spread fairly evenly across the genome, one SNP approximately every 100 bp (SSR may be 1 in 10,000 bp)

•May be located in the coding (cSNP)or non-coding regions

•May have a direct functional effect or may be closely linked to functional marker

SNPs in DNA Sequence

Base substitutions: Transition A/G, C/T Transversion A/C, A/T, G/C, G/T Indels: Insertion and deletions of 1-3 nucleotides VNTRs : polynucletide repeats

2. Molecular Origin of SNPs

Why are SNPs bi-allelic?

1. Low probability of spontaneous point mutations (10-6)

2. High probability of base transition as compared to base transversion

1. Re-sequencing of specific loci from different

varieties of a species.

2. Alignment of ESTs or RNA Sequence data

obtained from different varieties

3. Genomic clones (BAC, Plasmid) sequence

overlaps

4. Genome sequences from different varieties of

a species

3. SNP Discovery

SNPs in the BADH1 gene of rice

SNPs by Sequencing

Genotyping by direct sequencing is

expensive because it is low throughput

4. SNP Genotyping Assays and Platforms

ApekI sites PstI sites

GBS 96-plex Protocol (cont.)

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1. Plate DNA & adapter pair

4. PoolDNAs

5. PCR

Primers

2. Digest DNA with RE3. Ligate adapters

(may be done simultaneously)

ApeKI (5 base-cutter)

6. Evaluate fragment sizes

Clean-up

GBS 96-plex Protocol (cont.)

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1. Plate DNA & adapter pair

4. PoolDNAs

5. PCR

Primers

2. Digest DNA with RE3. Ligate adapters

(may be done simultaneously)

ApeKI (5 base-cutter)

6. Evaluate fragment sizes

Clean-up

GBS 96-plex Protocol (cont.)

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1. Plate DNA & adapter pair

4. PoolDNAs

5. PCR

Primers

2. Digest DNA with RE3. Ligate adapters

(may be done simultaneously)

ApeKI (5 base-cutter)

6. Evaluate fragment sizes

Clean-up

Sample DNA

A/G

GAA

G

GG

AA

AA

AA

AAGGAGA

AAG

G

Sample digestion, barcode and Illumina F and R adapters

Reference sequence

Step 1 Sample digestion using enzyme

Step 2 Barcode adapter ligation

Step 3 Pooling of samples, PCR amplification and

sequencing

Step 4 DNA sequences aligned to reference genome, sorted by barcodes and

SNPs called.

DNA

barcode

Universal

forward

primer

Digestion

site Digestion

site

Pooling PCR Illumina sequencing

Sequence sorting (bioinformatics)

SNP Genotyping by Sequencing (GBS)

Life Technologies Ion AmpliSeqTM Workflow

1. SNP by Direct hybridization

• Dot blot (genomic DNA or PCR product on slide, probe in mobile phase)

• Reverse dot blot [ASO (allele specific oligos) probes on slide]

e.g. 1. Microarray (Affyamtrix, Perlgene technologis)

2. Illumina ‘Golden gate’ and ‘Infinium’ technologies

Affymetrix SNP

Mapping

SNP GeneChips

10K

50K (x2 = 100K)

250K (x2 = 500K)

SNP Genotyping by Illumina’s

GoldenGate™ Assay

1. Allele Specific Extension and

Ligation

2. PCR Amplification

3. Hybridization to the Universal Sentrix® Array Matrix

A G

illumiCode’ Address Allele Specific Extension & Ligation

Universal

PCR Sequence 1

Universal

PCR Sequence 2

Universal

PCR Sequence 3’

Allele Specific Extension and

Ligation

Genomic DNA [T/C] Ligase [T/A] Polymerase

Custom Oligo Pool All (OPA)

96-1,536 SNPs multiplexed

Total oligos in reaction – 288-4,608

A illumiCode #561 Amplification Template

PCR with Common Primers

PCR Amplification

Cy3 Universal

Primer 1

Cy5 Universal

Primer 2

Universal

Primer P3

/\/\/\/

/\/\/\/

/\/\/\/

illumiCode

#561

illumiCode

#217

illumiCode

#1024

Hybridization to Sentrix® Array Matrix

/\/\/\/

/\/\/\/

A/A G/G C/T

SNP #561 SNP #217 SNP #1024

Sentrix® Array Matrix

10 mm 1.5 mm 400 mm

The Illumina BeadStation 500G permits high throughput analysis of

thousands of SNP DNA markers in hundreds of genotypes in less than

one week.

1 2 S e c t io n s

(m a x 2 8 8 K b e a d ty p e s , 1 4 4 K lo c i)> 8 9 0 ,0 0 0 fe a tu re s

p e r s e c t io n

A v e ra g e 3 0 fo ld

re d u n d a n c y

1 2 S e c t io n s

(m a x 2 8 8 K b e a d ty p e s , 1 4 4 K lo c i)> 8 9 0 ,0 0 0 fe a tu re s

p e r s e c t io n

A v e ra g e 3 0 fo ld

re d u n d a n c y

Illumina Infinium Assay

SNP BeadArrays

100K exon-centric

300K HapTag

13K non-synon coding*

550K* HapTag

*pending

Axiom® : Next Generation Genotyping & Expression Soln.

GeneTitanTM Instrument

Axiom Analysis Suite The Complete Solution

Kitted Arrays & Reagents

World’s First Hands Free Microarray Platform

Gene Titan: Powered by Axiom®

Hybridization Oven

Wash A, Wash B, Water, Waste

6 trays to position plates

Trash for covers

Wash A & B bath

Door to access the scanner

GeneTitan: the Inside

Salient Features

•Platform-Hands Free Automated processing platform •Fully Automated Genotype Assignment − automated genotype-calling algorithm supports diploid and polyploid genomes •Flexible Throughput – 16,24, 48, 96- and 384-array layouts for multiple applications ranging from Expression, SNP discovery to routine testing • Guaranteed Precision – No SNP dropouts or synthesis failures; photolithographic templates enable 100% of designed markers to be included on the array • Interfering SNP tolerance-10 bp for haploid, 20 for diploid and 30 bp for any polyploidy • Multiple Species Format- Flexibility to customize multiple species on the same array; maximum utilization of resources • Low minimums – Customizable arrays for as few as 480 samples; small repeat orders •Consistent supply – Designs never expire; access to the same array content for as long as your research necessitates

Axiom® background terminology

– Two colors/channels • AT channel: ligated A or T nucleotides are labeled with APC and emits

at 660nm

• GC channel: ligated G or C nucleotides are labeled with PE and emits at 578nm

• Terminology – non-AT/CG SNPs: SNPs that can be distinguished due to a different dye

label, e.g. SNPs that are either A or G, T or C, A or C etc.

– AT/CG SNPs: SNPs that are targeted by the same dye, e.g. SNPs that are either A or T

– Indel: known nucleotide deletions or insertions

Highly reproducible & legendary Photolithography

Array with probes specific to genomic position

DNA amplification, fragmentation and hybridization

Hybridize solution probes (9-mer) with specific attached hapten

DNA ligase to covalently bind the correct base and signal amplification

Everything that has not been ligated is washed away

Axiom® Chemistry Overview

Genomic DNA target (G)

30-mer on array

CNNNNNNNN TNNNNNNNN

Solution probes Two-color labeling

GNNNNNNNN

ANNNNNNNN

Ligation Event

Target Preparation Process Overview- bench

Mix with precipitation reagents

incubate: @-20C overnight

Centrifuge and dry the pellet

Resuspension and QC

Store @-20oC

Store @-20oC

Setup DNA amplification

Fragmentation

22-24 hour incubation @37oC

OR

OR

Day 1

Day 2

Day 3

Axiom processing on GeneTitan MC

Hybridization: 48oC, 23.5 hours

Post hyb wash: Wash A : RT 6min Wash B : 39oC, 25min Wash B: RT, 4min

Ligation: RT, 2 hr

Post Lig wash: Mol. grade H2O 50oC, 20min; RT, 4 min;

Stain and wash: RT., 10min/layer;

6 min wash A between each layer

Stablization: RT, 15min;

Holding: RT

Fluidics: ~5 hours

scan Scanning ~7.5hours

QC: Genomic DNA • Source of gDNA:

– Multiple sample source

• Amount of gDNA: – Input of 200ng: 20ul at 10ng/ul

• Must be of high quality, and free of contaminants – 260/280 ratio 1.8 – 2.0 and 260/230 ratio >1.5

– Assess the integrity of the DNA using a 1% agarose gel (~90% of the DNA should be greater than 10Kb)

25bp-

125bp-

25bp-

125bp-

4% E-gel: DNA should be between 25 – 125bp

Look for uniformity (size, yield) across samples

QC: Fragmentation

No effect of Secondary Polymorphism

Axiom tolerates a single base-pair mismatch outside of 10b window from the SNP interrogation site

5

ACCGTCTACGAAATATCTTTCACGTTGTCGTATAA ~

5

3

TNNNNNNNN-Hapten1

CNNNNNNNN-Hapten2

[A/G]

TTTATAGAAAGTGTAGAGCATATT

Mismatch outside of 10b window

•Often with highly polymorphic crop like crop • Thus high conversion rate of SNP tiled on the Array • Won’t miss the SNP of interest to tile on the array due to interfering SNPs

2. Generation and separation

of allele-specific products

PCR-RFLP, SNP to CAPS

SSCP (single strand conformational polymrphism, non-denaturing

gel), DGGE (denaturing gradient),

DHPLC (denaturation -reassociation, ion-exchange chromatography

mismatch pairing)

Capillary electrophoresis sequencers,

MALDI-TOF

Theory of SNuPe

ddATP*

ddCTP*

ddGTP*

ddTTP*

SNuPe = Single Nucleotide Primer extension • also called SBE (Single Base Extension), minisequencing

A

G

5’

3’

3’

5’

Unlabeled primer

A

G

5’

3’

3’

5’ T*

C*

+ Thermo Sequenase I SNuPe premix

C o m p a n y C o n f id e n t ia l

S N u P e F ra g m e n ts

• e n d -p ro d u c t is a p r im e r + a d y e - la b e le d te rm in a to r

• sm a ll f ra g m e n t o f D N A (~ 1 8 to 2 6 b a se s )

• re a lly ju s t s e q u e n c in g o n e b a se

5 ’ 3 ’T *

C *

T /T

h o m o z y g o te

C /T

h e te ro z y g o te

C /C

h o m o z y g o te

S N u P e p ro d u c ts

ru n o n th e

M e g a B A C E

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

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 TCGAAATGCATGCGCGTGATT TTACGTACGCG TTACGTACGCG

TCGAAATGCATGCGCATGATT TCGAAATGCATGCGCGTGATT TTACGTACGCGT 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

0

20

40

60

80

100

120

4300 4400 4500 4600 4700 4800 4900 5000

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

ZipCode1 GER

C G

1. OLA

2. Clean-up (removes unligated probes)

ASOY

ASOX A

G

ZipCode2

(Homozygote shown in this case)

P A

Universal PCR Priming site

GER LSO P

G

gDNA

Target C

GER

ABI SNPlex assay

3. Multiplexed Universal PCR

Univ. PCR Primer

Biotin

4. Capture

6. ZipChute™ Probe Hybridization

G

G

C (Streptavidin)

Plate

G Plate

G Plate

Univ. PCR Primer

Beckman SNPstream assay

Tag SNP primer

3’ 5’

PCR target

SNP site

Labeled terminating NTP

5’ 3’

SNP Primer

Tag complement for

Hybridization capture

X 12 or 48

KASPAR Assay

Genotyping by Sequencing (GBS)

1. Whole genome re-sequencing a. Physical shearing of genomic DNA b. Shearing by frequent cutter nucleases 2. Reduced representation sequencing a. RAD (restriction site associated DNA) sequencing b. dd RAD re-sequencing c. Targeted amplicon re-sequencing

5. Applications of SNP

Applications of SNP

1. Preparation of high density linkage maps

2. Diagnosis of predisposition to genetic diseases (animals)

3. Diversity analysis and fingerprinting of genotypes

4. Evolutionary studies based on synonymous substitution

rates in orthologous genes present in the related species

5. Association studies between SNP/ haplotypes and

phenotypic traits-linkage disequilibrium (LD) studies

6. Markers assisted selection (molecular breeding) using high

throughput SNP genotyping

Mapping Population: ♀ Pusa Dwarf X ♂ HDM04-1 Trait Pusa Dwarf HDM04-1

Plant height (cm) 88 118

No. of primary branches/plant

20 5

No. of pods/plant 120 24

Days to Flowering 106 65

Days to Maturity 158 116

Growth habit Determinate Indeterminate

Mapping of Plant type Traits in Pigeonpea

Pusa Dwarf HDM04-1

QTL Mapping for Plant Type and Earliness

57

S. No. Reference

Gene

Category

No. of genes in

Reference set

No. of

Identified

raw SNPs

No. of genes

included in the

Chip

Number

of SNPs

in the

Chip

Number of

SNP per

gene in the

Chip

1 AGCP 192 13,979 161 1,697 10.5

2 SCP 10,064 430,380 4,317 24,876 5.8

3 CSCSP 5,899 130,439 4,326 27,426 6.3

4 DRDRP 874 48,387 747 7,375 9.9

5 MCP 96 22,477 80 679 8.5

TOTAL 17,125 645,662 9,631 62,053 6.4

• 56 % of the reference genes incorporated in the chip

• Total 62, 053 SNP loci have been included in the chip

• Whole Pipeline has been automated and standardized

Development of a 62K genic-SNP Chip for Pigeonpea

•Seeds of 96 RILs and 96 Varieties from pigeonpea were used for validation.

•Genomic DNA was isolated and chip validation is completed

Fig. RILs population grown in Phytotron Table. Snapshot of Completed Pigenopea 62 K chip (RILs) successful run on Gene Titan

Pigeonpea 62K SNP Chip validation using Gene Titan & Axiom Analysis Suite 1.1.1

Fig. Cluster Plot of validated SNP Loci AX-123666045

Summary of validated 96 RILs population using

62 K SNP chip

CSCSP-11300_54080.0CSCSP-11300_1067 CSCSP-11300_5425CSCSP-11300_210

1.3

SCP-10822_389 CSCSP-10821_2833CSCSP-11300_4194 CSCSP-10821_2107CSCSP-10821_2286 CSCSP-11300_7139CSCSP-11300_2462 CSCSP-3727_2734CSCSP-11300_3839 CSCSP-3727_999SCP-10822_94 SCP-10822_587

1.4

CSCSP-3727_2973 CSCSP-10821_285CSCSP-10196_898

1.5

CSCSP-5170_3552.2CSCSP-5170_3312.6CSCSP-11300_54662.8CSCSP-15823_28875.2CSCSP-7119_27235.3SCP-12200_51129.0CSCSP-4778_35910.1SCP-5413_243 SCP-13167_10010.8CSCSP-5340_300610.9CSCSP-12451_1336611.1SCP-13553_27711.4CSCSP-5102_221811.8SCP-5993_59116.1DRDRP-363_38316.9CSCSP-6340_303917.6CSCSP-5169_131618.0CSCSP-6436_120119.7CSCSP-5169_116720.2CSCSP-5177_206623.1SCP-5055_77723.7SCP-5492_68323.9SCP-5492_48726.7DRDRP-596_115128.8SCP-13515_21329.1DRDRP-596_121729.3CSCSP-13663_173330.1CSCSP-10309_260935.5CSCSP-3538_5101 CSCSP-10309_3443SCP-9955_334

36.5

CSCSP-10309_816 CSCSP-5290_693CSCSP-3538_5865

36.6

CSCSP-2659_27137.3CSCSP-3538_398938.3SCP-5032_373238.7CSCSP-5608_112638.9SCP-6383_251039.0SCP-6383_1119 CSCSP-3538_208639.2SCP-9607_73341.1CSCSP-6545_21941.4CSCSP-1876_117641.6SCP-9601_124946.0CSCSP-12789_169848.6SCP-9601_83149.7SCP-1712_481 SCP-9601_89749.9SCP-9601_60450.2SCP-1716_294350.9SCP-9601_101551.0SCP-9601_106 SCP-9601_49251.1SCP-9601_114951.2SCP-9601_40551.7CSCSP-1624_469953.3CSCSP-1624_490253.8CSCSP-1624_4666 CSCSP-1624_5563CSCSP-1624_905

53.9

CSCSP-1624_42554.0CSCSP-1624_313354.4CSCSP-7144_127955.6CSCSP-13135_431662.1CSCSP-12592_54264.1SCP-12593_35564.4SCP-12799_1059965.0SCP-6272_52365.9CSCSP-3002_49367.5CSCSP-13135_126368.5CSCSP-11776_165368.9CSCSP-11776_744769.0CSCSP-11776_922069.1CSCSP-4373_95973.9AGCP-100_18976.5CSCSP-14850_187576.9CSCSP-4373_131377.4SCP-3348_142 DRDRP-249_81482.5DRDRP-703_83882.9DRDRP-703_1054 DRDRP-370_45985.7DRDRP-703_253 CSCSP-15954_3567DRDRP-703_745 DRDRP-703_1575DRDRP-370_558 DRDRP-690_398DRDRP-690_229

85.8

DRDRP-703_161385.9DRDRP-703_185886.0CSCSP-7436_1046 CSCSP-7436_175986.5DRDRP-703_266486.9DRDRP-703_2649 DRDRP-370_52287.0DRDRP-690_103387.1CSCSP-7436_255487.6CSCSP-7436_79687.9DRDRP-690_107188.5DRDRP-690_130789.0DRDRP-690_102090.0AGCP-100_160794.1CSCSP-10999_363594.4CSCSP-14100_349297.9CSCSP-191_105101.4CSCSP-191_854102.5CSCSP-191_273104.9CSCSP-191_262105.5SCP-13688_574107.4SCP-2938_605109.5

1

CSCSP-6260_41170.0CSCSP-6260_48420.1CSCSP-6260_49020.2CSCSP-14312_77790.4CSCSP-6260_49531.7CSCSP-6260_13742.2SCP-8809_3065.6DRDRP-250_11106.6DRDRP-250_835 DRDRP-337_7337.5SCP-4320_79178.1SCP-4320_77448.3DRDRP-250_17878.6DRDRP-250_14639.3DRDRP-250_157710.5CSCSP-7426_234311.2DRDRP-250_19312.3DRDRP-250_95613.0DRDRP-250_224 DRDRP-250_725SCP-14005_2588

13.2

DRDRP-337_70513.3DRDRP-250_42313.5DRDRP-337_551 CSCSP-4005_2678CSCSP-7168_4211 DRDRP-250_278

13.9

DRDRP-337_423 DRDRP-250_1047DRDRP-337_569

14.4

DRDRP-250_1034 DRDRP-250_746DRDRP-250_1875 DRDRP-250_1926

14.5

DRDRP-337_148714.7DRDRP-250_59115.2CSCSP-13358_8319.9SCP-11093_64424.6CSCSP-6182_748527.3CSCSP-6182_510828.4CSCSP-6182_1104829.2CSCSP-1843_446129.7SCP-6183_353829.8CSCSP-6182_607530.0CSCSP-1843_178430.1CSCSP-6182_985830.2SCP-6183_345 CSCSP-1843_2326SCP-6183_136 CSCSP-13682_1988CSCSP-6184_1551 CSCSP-1843_4268CSCSP-1843_1709 CSCSP-6182_6195CSCSP-1843_5352 CSCSP-6182_9892

30.3

CSCSP-1843_1595 CSCSP-6182_9508SCP-6183_1632 CSCSP-1843_1156CSCSP-13682_1548 CSCSP-6182_10939CSCSP-6182_2453 CSCSP-6182_12020CSCSP-6184_1170 CSCSP-6182_4874CSCSP-1843_3900 CSCSP-6182_2585CSCSP-6182_11016 CSCSP-1843_4983CSCSP-6182_9808 CSCSP-6182_82SCP-6183_2902 CSCSP-6182_8194SCP-5568_622 CSCSP-6182_10388CSCSP-6184_345 CSCSP-6182_786CSCSP-6182_13692 CSCSP-12634_4677CSCSP-6182_3463 CSCSP-6182_6297CSCSP-6182_4738 CSCSP-6182_12680CSCSP-1843_3387 CSCSP-1843_2312CSCSP-1843_4747 CSCSP-1843_1565SCP-6183_786 SCP-6183_1962SCP-6183_548

30.4

SCP-6183_273 CSCSP-6182_10016CSCSP-6182_690

30.5

CSCSP-6182_8179 SCP-6183_187930.6CSCSP-1843_243031.4CSCSP-6182_533031.7CSCSP-4680_538431.9CSCSP-6182_623632.9CSCSP-14453_190735.7SCP-7752_13137.2SCP-5568_60142.9

2

DRDRP-530_18140.0SCP-14292_2321.4DRDRP-84_2322.9SCP-10986_2685.4SCP-14292_1796.3DRDRP-851_3038.1SCP-11337_239514.7SCP-11337_194414.9SCP-5530_231119.6CSCSP-4063_576220.2SCP-6170_85420.4DRDRP-138_89220.9DRDRP-195_138121.1CSCSP-4063_372321.4CSCSP-1570_311621.7DRDRP-414_33021.8DRDRP-320_153521.9DRDRP-830_246522.0DRDRP-414_83822.1CSCSP-1570_2694 DRDRP-830_344322.2CSCSP-4063_3722.3CSCSP-4063_3455 DRDRP-320_1773DRDRP-830_2666 CSCSP-4063_1429DRDRP-195_1097 DRDRP-830_3034DRDRP-830_3460 DRDRP-320_1837SCP-14389_307 DRDRP-830_4453DRDRP-320_4920 SCP-14389_1128DRDRP-195_1262 CSCSP-4063_3175SCP-14389_845 DRDRP-320_4693SCP-14389_409 CSCSP-4063_3734SCP-14389_277 SCP-14389_876DRDRP-830_2734 DRDRP-830_333DRDRP-830_4434 DRDRP-320_426CSCSP-1570_3062 DRDRP-320_408CSCSP-4063_4138 DRDRP-830_1868DRDRP-320_4735 DRDRP-414_299DRDRP-830_3379 DRDRP-320_340SCP-14389_1093 CSCSP-1546_558DRDRP-593_479

22.8

DRDRP-830_3783 DRDRP-414_907CSCSP-1570_2510 SCP-14389_865CSCSP-1546_618 DRDRP-320_4878DRDRP-138_461 DRDRP-195_431SCP-10211_837

22.9

CSCSP-1570_358123.3DRDRP-830_415123.4DRDRP-138_1359 DRDRP-830_4056CSCSP-1570_3299

23.6

SCP-14389_118923.8DRDRP-830_228423.9DRDRP-320_438224.1DRDRP-830_299024.7SCP-9593_25725.2CSCSP-7024_63433.8CSCSP-6163_184534.0SCP-2894_91035.7DRDRP-748_104438.9DRDRP-343_1005 DRDRP-748_90939.1SCP-13333_33740.3SCP-13333_94040.6DRDRP-42_389541.1CSCSP-11663_6341.3SCP-12265_387 SCP-12265_312CSCSP-14824_491

41.4

CSCSP-14824_1410 SCP-12265_29441.5SCP-12265_9542.4AGCP-178_240243.4SCP-11683_62646.3CSCSP-7712_374047.6DRDRP-411_183248.6SCP-9748_210048.8SCP-14103_238454.0SCP-14736_494 CSCSP-9805_53854.7SCP-6408_236154.8SCP-14736_277654.9SCP-5258_131255.2SCP-4099_209655.3CSCSP-8819_628 CSCSP-8819_992SCP-5258_66

55.4

SCP-14103_72455.5SCP-14103_86956.5SCP-12160_25157.8CSCSP-10988_11859.4SCP-9748_235461.5

3

CSCSP-11828_17100.0SCP-4820_3672.5DRDRP-410_14193.5CSCSP-6196_866.0CSCSP-6243_3776.7SCP-14541_2988.0CSCSP-6243_16818.6CSCSP-6243_39910.9AGCP-126_118 AGCP-126_246212.5AGCP-126_29012.8SCP-10151_47013.6CSCSP-6700_1512 CSCSP-6700_172818.1CSCSP-6700_203418.9CSCSP-6700_193219.3CSCSP-6700_2670 CSCSP-6700_1792CSCSP-6700_2739 CSCSP-6700_2651CSCSP-6700_2178 CSCSP-6700_2157CSCSP-6700_568

19.5

CSCSP-14209_92021.6CSCSP-14209_86321.7CSCSP-14209_61022.2SCP-1567_31227.6CSCSP-12784_470030.0CSCSP-12784_476030.3CSCSP-5610_42531.7CSCSP-14069_153132.2SCP-9592_758835.4SCP-4174_1231637.8AGCP-100_398239.0SCP-9592_7115 CSCSP-14921_2441SCP-9592_4513

39.5

CSCSP-13484_6930 CSCSP-13484_1427CSCSP-13484_8819 CSCSP-13484_2489CSCSP-13484_4551 CSCSP-3007_121CSCSP-13484_6669

39.6

CSCSP-13484_1484 CSCSP-13484_228739.7CSCSP-14921_248839.8CSCSP-13484_163839.9CSCSP-2896_26240.7CSCSP-14921_457941.1CSCSP-3007_30242.0SCP-6384_350642.4CSCSP-14982_27342.7SCP-6384_112442.8CSCSP-7031_52343.5CSCSP-1907_506543.7CSCSP-14982_674 CSCSP-1907_489143.8SCP-3468_74844.8SCP-3468_64645.7AGCP-119_45346.8AGCP-119_186447.0CSCSP-1641_622449.7CSCSP-10060_85750.3SCP-12684_5451.9CSCSP-4399_161853.1SCP-4087_49053.3CSCSP-6819_3550 SCP-13775_279454.1CSCSP-6819_351654.3SCP-13976_14354.4SCP-222_40354.7SCP-12663_478456.0DRDRP-305_560256.9CSCSP-3708_51358.2SCP-13413_41958.7CSCSP-4425_66861.8CSCSP-10917_2277 CSCSP-10917_68464.1CSCSP-10917_793 SCP-4746_74864.2CSCSP-10917_146065.0CSCSP-11684_485366.9CSCSP-11684_14132 CSCSP-11684_1095767.4CSCSP-4425_454367.5SCP-13033_83569.3CSCSP-4425_105769.8CSCSP-13242_127769.9CSCSP-4425_407771.9SCP-13033_824 CSCSP-11684_1193472.4CSCSP-15304_629 CSCSP-3677_123878.8CSCSP-4483_54379.2CSCSP-15304_1709 CSCSP-15304_176379.4CSCSP-15304_1416 CSCSP-15304_114079.9CSCSP-15304_119980.0CSCSP-3677_1875 CSCSP-15304_124180.4SCP-4746_171981.1CSCSP-4483_94481.6CSCSP-3677_58082.9SCP-14218_72886.0

4

DRDRP-205_12960.0DRDRP-240_16450.9DRDRP-205_8241.9DRDRP-205_3265.3DRDRP-205_3496.6DRDRP-205_16387.9DRDRP-240_8048.6DRDRP-240_676 DRDRP-205_14018.7DRDRP-336_3259.2DRDRP-205_11189.8DRDRP-205_4589.9DRDRP-341_1753 DRDRP-205_50910.0DRDRP-336_171010.3DRDRP-205_113010.4DRDRP-720_283612.5CSCSP-10631_101113.1DRDRP-204_65814.0DRDRP-204_204 DRDRP-204_80314.3DRDRP-204_43914.4DRDRP-204_150415.3DRDRP-387_18515.7DRDRP-204_100016.3DRDRP-204_1482 DRDRP-204_143516.5DRDRP-720_273616.9CSCSP-7264_97317.2SCP-11466_187 DRDRP-204_45617.4DRDRP-204_134417.8DRDRP-204_1275 DRDRP-204_171817.9SCP-11466_43718.3CSCSP-6043_58918.9CSCSP-6043_127419.0CSCSP-7742_120919.1CSCSP-6043_222219.2CSCSP-10631_52019.4SCP-12356_183919.7DRDRP-386_149123.5CSCSP-7264_315324.3DRDRP-722_1221 DRDRP-722_96824.9DRDRP-386_209925.2DRDRP-748_36425.4DRDRP-386_144225.5DRDRP-722_22925.7DRDRP-722_1803 DRDRP-722_3030DRDRP-386_2267 DRDRP-386_780DRDRP-386_1884

25.8

DRDRP-386_2223 DRDRP-386_1471DRDRP-720_952 DRDRP-722_1372

25.9

DRDRP-386_994 DRDRP-722_210326.0DRDRP-386_51226.1DRDRP-386_43226.2CSCSP-9474_2524 CSCSP-4941_40326.3CSCSP-12446_4606 DRDRP-386_156926.5DRDRP-386_49426.6CSCSP-9474_261826.7SCP-11412_62027.0DRDRP-386_221127.6DRDRP-240_87928.2SCP-2911_44728.9DRDRP-722_249729.6DRDRP-720_131329.8DRDRP-720_2509 DRDRP-720_152330.0DRDRP-720_161630.3DRDRP-722_164630.4DRDRP-720_316530.7DRDRP-720_147034.2SCP-13780_50639.3SCP-13878_20145.4SCP-13878_188445.6CSCSP-15158_2506 CSCSP-15158_3189CSCSP-15158_3543

46.5

CSCSP-5713_1009 CSCSP-8812_43847.3DRDRP-47_1445 CSCSP-5713_2919CSCSP-5713_2953 CSCSP-5713_3252CSCSP-5713_1691 DRDRP-47_804CSCSP-5713_1835 CSCSP-5713_1732

47.4

CSCSP-5713_30947.5SCP-13878_178548.4CSCSP-4506_4443 CSCSP-4506_346648.5SCP-13878_1290 CSCSP-4506_198148.6CSCSP-15158_322549.8SCP-13878_186951.2DRDRP-512_65455.4DRDRP-32_254556.7

5

DRDRP-510_406 DRDRP-510_3910.0DRDRP-510_8420.1DRDRP-510_4690.2DRDRP-510_6290.4CSCSP-7393_27133.4SCP-15114_14784.4CSCSP-7393_12967.9CSCSP-7393_124910.1CSCSP-7393_307010.3CSCSP-7393_244210.4DRDRP-647_484410.7CSCSP-13667_45211.3CSCSP-13667_94911.6DRDRP-647_463911.9CSCSP-6895_4296 DRDRP-647_877DRDRP-647_3268

12.0

DRDRP-647_233012.8CSCSP-14820_443 CSCSP-14820_1151CSCSP-14820_1587 CSCSP-14820_463

16.0

CSCSP-5138_25616.9SCP-13077_2553 SCP-15114_69619.1SCP-15114_63119.2SCP-15114_1873 SCP-15114_108119.8SCP-15114_84721.7SCP-15114_15224.4CSCSP-7746_200727.0CSCSP-7746_199428.9SCP-1764_101830.7CSCSP-3731_393336.3SCP-1925_101638.4CSCSP-12048_91139.2SCP-11571_10039.9CSCSP-10898_199140.1SCP-10112_149641.2CSCSP-5407_156841.4SCP-10070_96241.6SCP-10112_180242.0SCP-10112_858 SCP-10112_1677SCP-10174_729 SCP-10112_1666SCP-10112_951 SCP-10112_1038

42.1

SCP-10198_72942.6SCP-10112_75245.3SCP-4003_122046.8SCP-12057_105147.0SCP-229_52449.2CSCSP-13527_184454.0CSCSP-3330_285254.3CSCSP-11703_136754.4CSCSP-12702_397259.0CSCSP-12702_1870 CSCSP-12702_511459.1CSCSP-12702_375559.5

6

SCP-5327_34720.0CSCSP-11017_26382.2CSCSP-10928_37984.4CSCSP-10928_21715.4CSCSP-10928_21125.5CSCSP-10928_36755.6MCP-51_31368.9SCP-5120_23310.3CSCSP-11200_224214.0CSCSP-6795_123816.9CSCSP-6795_4996 CSCSP-6795_1175317.6CSCSP-6795_9378 CSCSP-6795_394917.8CSCSP-6698_145417.9CSCSP-10697_74019.2CSCSP-10697_12720.2CSCSP-6795_206521.3CSCSP-11423_70223.1CSCSP-11200_204323.7CSCSP-11423_363224.3CSCSP-10697_114224.9CSCSP-3684_509725.9CSCSP-10697_625 CSCSP-3684_495526.0CSCSP-11849_203126.2CSCSP-1970_93026.4DRDRP-369_114026.7CSCSP-1542_1390 SCP-3210_29327.0CSCSP-1542_172727.1CSCSP-3684_382427.7CSCSP-11423_139228.3SCP-11630_114729.0SCP-11630_432 CSCSP-5643_9929.1CSCSP-3979_197429.6CSCSP-9960_1031 CSCSP-9960_1060SCP-11671_630

29.7

CSCSP-10697_39330.1SCP-3019_6830.9CSCSP-3979_171831.7CSCSP-3979_58732.3DRDRP-811_84333.3DRDRP-797_409 DRDRP-797_6933.8DRDRP-797_248 DRDRP-797_27533.9DRDRP-797_20034.1CSCSP-4897_130634.5SCP-11630_71135.8DRDRP-811_149636.3SCP-11630_90536.7

7

CSCSP-13756_7360.0SCP-2040_13384.0CSCSP-12087_18846.2CSCSP-12170_30618.2SCP-9783_14312.2CSCSP-11897_93912.9SCP-9783_25013.3CSCSP-11897_245413.7CSCSP-11897_20814.4CSCSP-11897_3492 SCP-9783_44815.0CSCSP-11897_35915.6SCP-10172_1809 SCP-9560_1431SCP-9560_682 SCP-10172_153SCP-9560_1578

15.7

SCP-9560_332 SCP-9560_1493CSCSP-13306_4887

15.8

SCP-10172_47616.1CSCSP-12170_250116.2SCP-9560_62817.3SCP-10172_125918.3SCP-10172_196619.5SCP-9677_118126.6CSCSP-13110_100128.5CSCSP-14791_100628.9SCP-14628_50129.0SCP-3809_31030.9SCP-2947_156932.4CSCSP-11029_52433.0SCP-9702_71934.1SCP-9660_71435.6SCP-2947_172036.6SCP-12705_100636.8SCP-2947_7736.9SCP-2947_162937.1CSCSP-1658_48238.8CSCSP-15159_18140.3SCP-2947_177642.6SCP-9660_6442.7SCP-13732_69944.5SCP-13732_73344.8SCP-7705_4806 SCP-7705_470245.0SCP-7705_432845.1CSCSP-14886_97546.3SCP-13732_101949.1CSCSP-13263_17749.8CSCSP-11457_166751.9SCP-14707_33055.3CSCSP-12276_562857.8

8

CSCSP-10659_7830.0CSCSP-13285_4131.0CSCSP-13285_4612.6CSCSP-11018_84073.8CSCSP-11018_54224.4CSCSP-11018_2815.2CSCSP-10659_9707.2CSCSP-10659_10157.3CSCSP-10659_10539.0SCP-15353_130111.1SCP-4028_217511.2CSCSP-11018_3522 CSCSP-11018_2567CSCSP-11018_4756

11.3

CSCSP-11018_6164 CSCSP-11018_183011.4SCP-4028_264711.5CSCSP-11018_594412.1SCP-4028_445312.9CSCSP-11018_351113.3CSCSP-11018_940 CSCSP-11018_1532CSCSP-11018_4784

13.4

CSCSP-11018_507513.5CSCSP-11018_1025 CSCSP-11018_73413.6CSCSP-11018_7444 CSCSP-11018_814CSCSP-11018_903

14.2

CSCSP-6256_358423.9SCP-5142_172124.0CSCSP-6256_116024.3CSCSP-1833_132325.1CSCSP-1833_113025.6CSCSP-4518_432832.2CSCSP-4181_10334.4CSCSP-5490_106134.5CSCSP-4181_434234.7CSCSP-5488_76234.9CSCSP-4181_423735.0CSCSP-5488_72335.1CSCSP-5490_122136.2CSCSP-4181_213036.8CSCSP-4518_46338.4CSCSP-5508_74543.0

9

CSCSP-6821_45040.0CSCSP-6821_56521.8CSCSP-6821_1652.6CSCSP-6821_10532.9CSCSP-6821_6838 CSCSP-15851_32063.0CSCSP-11871_75110.8CSCSP-4316_200217.6SCP-14083_323922.1SCP-10135_46022.4SCP-14491_36724.8DRDRP-806_1066 CSCSP-5191_35626.3SCP-9779_148528.6CSCSP-5042_78931.0DRDRP-405_256431.1SCP-6017_12031.3DRDRP-405_274631.9SCP-3935_46432.6SCP-3935_65434.0SCP-3935_68535.3SCP-12318_6137.3CSCSP-6101_18238.9CSCSP-1702_436940.0SCP-3337_65741.4SCP-13513_72143.2SCP-15677_14543.9CSCSP-6101_76744.2CSCSP-13157_4647.8SCP-5824_37248.8DRDRP-827_234552.0DRDRP-827_250352.4DRDRP-858_1166 DRDRP-827_1099DRDRP-827_1084

53.1

DRDRP-348_189653.2DRDRP-348_16753.9DRDRP-827_123455.2DRDRP-477_94455.3DRDRP-858_141759.2CSCSP-6022_251562.1DRDRP-858_205662.3SCP-7381_1057 CSCSP-7473_39662.8SCP-243_448 SCP-243_28363.0SCP-3154_52963.1CSCSP-6022_1437 CSCSP-6022_223963.2SCP-3154_24164.7CSCSP-7473_68168.5CSCSP-4063_570372.0CSCSP-3912_69974.2DRDRP-390_22277.5

10

SCP-13888_15920.0AGCP-61_431013.0CSCSP-4948_87715.7CSCSP-14178_2316 CSCSP-6588_24416.8CSCSP-14178_2523 CSCSP-6588_223SCP-3846_1291 CSCSP-12542_534CSCSP-3040_460 SCP-6970_647CSCSP-3040_1024

16.9

SCP-6970_614 CSCSP-12542_36917.0CSCSP-1813_215925.3CSCSP-11654_101829.2SCP-6104_36631.1SCP-5184_305531.3CSCSP-13521_95634.4CSCSP-13030_182 CSCSP-13521_5834CSCSP-13521_7238

34.5

CSCSP-12742_36436.0SCP-5184_400137.9CSCSP-11061_297541.4DRDRP-670_250543.1SCP-12347_94647.2

11

LG1 LG2 LG3 LG11 LG4 LG5 LG6 LG7 LG8 LG9 LG10

A Linkage Map of Pigeonpea Pusa Dwarf/H2001-4 RILs with 858 SNP Markers

2. In diagnosis for genetic predispositions

against major genetic diseases

3. Application in MAS

Single Nucleotide Polymorphisms (SNPs) are the most abundant sequence variations encountered in

most genomes (Cho et al., 1999; Griffin and Smith, 2000). Various large-scale discovery projects are

currently aiming at identifying SNPs from a broad range of organisms, including crop plants. The

abundance, ubiquity and interspersed nature of SNPs make them ideal candidates as molecular

markers for marker-assisted plant breeding.

While various SNP detection methods have been described (Landegren et al., 1998), one objective is

to identify a co-dominant and robust system amenable to multiplexing and automation to genotype

SNPs from plants at the seedling stage. Example is the development of Single Nucleotide Primer

Extension (SNuPE) assays enabling co-dominant genotyping of SNPs from small amounts of barley

tissue. The method was used to select barley seedlings carrying the mlo powdery mildew resistance

allele as well as superior alleles of β-amylase thermostability based on SNPs discovered within genes

encoding these traits in barley.

Sequenom SNP genotyping service

ALLELE MINING FOR BETAINE ALDEHYDE

DEHYDROGENASE 1 (badh1) GENE IN RICE

(Oryza sativa L.)

BADH ENZYME

The biological significance of BADH is that it catalyses synthesis of a common osmoprotectant glycine betaine.

In many bacteria, plants and animals the glycine betaine is synthesized in a two-step reaction, first enzyme choline oxydase (COD) converts choline to betaine aldehyde and then enzyme BADH catalyses second reaction converting betaine aldehyde to glycine betaine: COD BADH

Choline Betaine aldehyde Glycine betaine

Glycine betaine is a compatible organic solute which accumulates to high concentrations in cells in response to drought, salinity and low temperature stress to protect cell structure and molecular functions.

LIST OF RICE VARIETIES USED

M1 A B C D E F G H I J K L M N O

P Q M2 M3

Quality check and quantification of genomic DNA extracted from rice varieties

M1-M3= Different amounts of Lambda genomic DNA (100, 200, 300 ng)

A-Q = Rice varieties

Plant DNA Extraction

PCR Primers Designed for Amplification of badh1 Gene Fragments

Optimization of PCR annealing temperature for different primers using gradient PCR A, BAD1-2

B, BAD1-3.1

C, BAD1-3.2

A

B

C

45oC to 65oC M

M1 A B C D E F G H I J K L M N O P M2

PCR amplification of a segment of the badh1 gene using PCR primer BAD1-6

A. Jyoti, B. Kalanamak 3119, C. MI 48, D.Taipei 309, E. CSR 10, F. Pokkali,

G.Jaya, H. PusaNPT 11, I.CSR 27, J.Pusa 44 , K.Pusa 1121, L. Kalanamak

3131, M.CSR 36, N.Ratna, O.Red Triveni, P.Pusa 1342; M1 and M2 100 bp

ladder

Sequencing of the badh1 Gene Fragments

An overview of the sequence contigs of the badh1 gene of 16 rice varieties based on sequence reads obtained using 16 pair of primers, assembled using Phred/Phrap/Consed software)

Consed window showing location of one of the 20 SNPs discovered by sequencing of the badh1 gene fragments from 16 rice varieties

Location of PCR primers (reverse primer underlined) and 20 SNPs (highlighted yellow)

in the badh1 gene of rice. The gene has 15 exons (in bold) and 14 introns

Sequence of the pre-amplification primers (PCRP) and single nucleotide extension primers (UEP) for genotyping of 20 SNPs by Sequenom MassARRAY MALDI-TOF system

• DNA samples (384 no.) are mixed with a

matrix, spotted onto a MALDI plate and

loaded into the Mass Spectrometer.

• Each spot is subjected to pulses of nitrogen

laser (337nm) in vacuum, which vaporises

and ionises sample. Matrix absorbs most of

the laser energy, preventing degradation of

the sample, and allows ionisation of some of

the DNA substrate.

• Application of an electric field causes DNA

ions to enter flight tube and are accelerated

towards Mass detector.

• All ions gain same kinetic energy, so larger

ions take longer to reach detector.

• The variation in Time of Flight allows

separation based on size.

MALDI-TOF Mass Spectrometry

Pusa

NPT11

Pusa

1342

Genotyping of ‘badh1_S5’ SNP Using Sequenom MassARRAY

badh1_S5

Unextended

primer

badh1_S5

Primer

extended

with C

badh1_S5

Primer

extended

with T

Another

Primer

of the

Multiplex

SNP allele calls from Sequnom MassARRAY system

and their cross validation with sequence information

Overall

Validation

Success

was > 80%

Showing only part of the Table, Total 81 SNP calls were validated

Summary of the SNP alleles in 16 rice varieties

and reference variety Nipponbare at 20 different

positions in the badh1 gene

SNP: BADH1- S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 S11 S12 S13 S14 S15 S16 S17 S18 S19 S20

CONCLUSIONS

1. The badh1 gene in rice has at least 20 SNPs distributed over the entire

length of the 4500 bp gene

2. Frequency of SNPs was higher in introns (17 SNPs, 1/ 150 bp) as

compared to exons (3 SNPs, 1/ 500 bp)

3. Assays were designed for the 20 SNPs identified by sequencing and

validated for high throughput genotyping using Sequenom MassARRAY

system

4. Rice varieties could be grouped according to their origin based on the

20 SNPs of badh1 gene

5. There was no obvious association between the badh1 SNP pattern and

salt tolerance trait of the 16 rice varieties

6. More studies are needed involving badh2 and cmo genes of rice to see

if betaine-based salt tolerance is present in any rice variety

Authentication of basmati rice by DNA genotyping

• Micro-satellite markers • Single nucleotide polymorphisms

Authentication basmati rice using SNPs

• PCR based SNP genotyping • MALDI-ToF MS based SNP genotyping

Quantitative SNP assays

MALDI-TOF-based

DNA authentication of basmati rice

Sponsored by the FSA

Basmati rice varieties

Traditional breeds

Basmati 386

Basmati 370

Dehradun

Ranbir Basmati

Taraori (HBC-19)

Cross bred Basmati 385 Super Basmati Pusa Basmati 1 Kasturi Basmati 198

Common Adulterants Pusa 169 Sabarmati PR 106 Kali-much Lakra Saket 4 Parimal Sherbati Terricot

RICE VARIETY Basmati 370 Derhadun Kernal PK370 l Pak 385 Pusa 1 Sherbati Super Taraori

LINEAGE Pure Bred Pure Bred Pure BredPure 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 GA

ory-592 GG GG AA GG GG AA GG GG AA

ory-599 TC TC TC TC CC CC CC TC TC

ory-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 11 primers

Tilda D6 Asda D5 Kernal (a1)

A AA AA AA

B AA AA AA

C GG GG GG

D TT CC TT

E GG CC GG

F GG GG GG

G GA GA GA

H AA AA AA

I TC CC TC

J GG GG GG

K 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 2003

ory-122-1 GG GG GG

ory-58-2 AA AA AT

ory-262 GG GG CG

ory-S0186 TT TT CT

ory-S0157 GG GG CG

ory-S0153 GG CC GG

ory-462 AG AG AG

ory-592 GG AA GG

ory-599 CT CT CC

ory-624-1 GG GG CG

ory-668 AA AA AG

SNP analysis as a QC tool

S+T 2003

GG GG

AA AT

GG CG

TT CT

GG CG

GC GG

AG AG

GA GG

CT CC

GG CG

AA 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

Monitoring the Spread of Rice Varieties (IRRI)

Reference varieties (Breeder Seeds): India: 34 (STRASA), 16 (Q2V) Nepal: 34 (STRASA)

Field Samples: India: 1917 samples (Odisha, W. Bengal, Bihar, U.P)

Nepal: 605 samples

Reference Varieties

Nepal: Mithila, Bindeshwari, Radha4, Hardinath 1, Radha17, Rampur Masuli, NR1488, Masuli, Basmati(awnless) , NR 601-1-1-5, Sabitri, Janaki, CH45, Radha11, Ram Dhan, Sukha Dhan 6, Loktantra, NR1190, IR 77721-93-2-2, Kanchhi Masuli, Ghaiya 1, BW 306, Sona Masuli, Makwanpur 1, Tarahara 1, IR 83388-B-B-108-3, Sabha Mahsuri Sub-1, Sukha Dhan 1, SukhaDhan 3, NR274, Naveen, KalaNamak, Pusa 834, Swarna Sub1

India: R.Suwasini, Rajshree, Rajendra Mahsuri, Rajendra Kasturi, Prabhat, Sudha, Vaidehi, Turanta, Jyothi, MTU-1010, PR-118, HKR-127, Sarala , Savitri, Swarna Sub-1, Gayatri ,Varsha Dhan, Ketaki Joha, CR-1014, Moti, Naveen, Lunishree, Padmini, Pooja, Pusa6(B), Pusa6(A), RP Bio-226, NLR34449, MTU7029, Sampada (IET-19424), BPT3291, IR 68897A, Jarva(IET-15420), Vardhan(IET-18940), IR64, IR64-Sub1, HUR105, Sarjoo52, Pusa44, PR114, Ranjit, Bahadur, MTU1075, ADT39, ADT46, ADT45, Gayatri, Pooja, Pratiksha, RPbio 226, (HKR127, Sarla, Padmini, Pusa 6 (B) did not germinate)

Genomic DNA Isolation Workflow

5-6 mg leaf tissue from each seedling was used for grinding in

Tissue Lyser at a frequency of 22/sec for 4 min (Qiagen-2004

model).

DNA was isolated using NucleoMag®96 Plant Kit

(Macherey- Nagel) as per manufacturers guide lines

The whole DNA isolation was performed on

KINGFISHER FLEX platform (Thermo Scientific)

DNA was eluted in 80 µl TE buffer

Seeds germinated in trays and grown for 10-15 days

DNA was checked on 0.8%

agarose gel using λ uncut

DNA for reference

DNA Quality Check and Quantification

DNA quality was checked

on Nanodrop for OD

260/280 and 260/230 ratios

(1.8 to 2.0).

DNA was diluted to 10ng/µl

with low T.E buffer

AmpliSeq 49 target regions in the Rice genome

Chromosome no.

Target regions

IR 64

IR 64 Sub1

Visualization of 2 SNPs in the Sub1A gene

Target region with two SNPs in Sub1 locus of IR 64 and IR 64 Sub1

IR 64 IR 64 Sub1

Functional SNPs in Sub1 locus of IR 64 and IR 64 Sub1

A Section of the SNPs Variant Matrix 120 SNPs 96 Sample

AmpliSeq SNP Assay - Run-Summary

Data Analysis- 50 Reference Samples

Data Analysis- 96 Samples (Reference and Field Samples

Cassava Monitoring Survey for Nigeria (CMS)

The role of DNA fingerprinting in accurate variety identification

March 17-18, Dar es Salaam, Tanzania

Case%Study:%use%of%genomic%tools%in%the%next%genera7on%cassava%project!

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Pantone 453 C

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Logo Nextgen Cassava Color palette

Primary color palette

Black and white version Reverse version

Extended color palette

Group 1 deliberations

• DNA fingerprinting procedure/methodology

• Variety identification analysis pipeline

• Field sampling logistics and sample tracking

• SPIA project in Ghana as a case study

• Metadata and other field data (areas)

Sample collection, preservation and DNA extraction

GBS genotyping process overview

Data analysis

Cornell Genomic Diversity Facility

IITA-Ibadan

Varietal identification analysis pipeline

1. Discover genetically identical accessions (i.e. samples representing same clone)

2. Determine genotype identity and status

Requires a comprehensive ‘library’ of known improved varieties and landraces

3. Estimate clone ancestries to uncover contribution of improved varieties in the genetic make-up of new cultivars

Discover genetically identical accessions

• Pairwise genetic distance between all accessions

• Empirically determine the distance threshold for declaring two accessions as genetically identical (same clone/variety)

• Cluster analysis (Hierarchical Cluster method)

• Ancestry estimation (resolving cases of admixtures/hybrids)

Determining genotype identity and status

• Matching accessions from farmers’ field to a DNA fingerprint database of “library” of known improved varieties and landraces.

• Such library already exists, thanks on ongoing RTB and NEXTGEN Cassava project.

• > 4000 accessions already genotyped at high-density using same GBS method at Cornell

Field sampling logistics

• One team for each region consisting of 5 enumerators, 2 cassava specialists, 1 supervisor (QC) and 1 local extension agent

• Cover a minimum of one village per day

• Collect 3x more leaf samples incase we need to re-extract.

• If possible, collect stakes for planting in Ibadan (field evaluations).

Ensuring chain of custody of sample ID from HH to field

• Thorough training of enumerators and field crew • Sample information on sampling tube:

– Region ID: (1, 2, 3) – LGA ID: (1, 2, 3, 4, 5, 6 ..... 25) – Enumeration Area ID: (1,2,3,4,5) – HH ID: (1,2,3,4,5) – Field ID? Plot ID? – Variety ID (1,2,3.....n) – Variety Name (e.g. “Agric”, “Oko – iyawo”) – GPS co-ordinate

• Each DNA tube has pre-printed barcode numbers that will be typed in the questionnaire and must correspond to specific variety in the concerned HH.

Ghana casa study Partners: MSU/IITA/CSIR-CRI/SPIA

• Collected 917 accessions from 495 farms in 3 regions

• ‘Library’ of 67 clones from CSIR-CRI

• Genotyping outsourced to Genomic Diversity Facility of Cornell

• Generated more than 50K SNPs

182 unique names but most are rare (<5 occurrences)

Genetic distance threshold for declaring two accessions as identical Histogram of RTB.IBD

Distance (1−PSA)

Fre

qu

en

cy

0.00 0.05 0.10 0.15 0.20 0.25

05

00

00

10

00

00

15

00

00

Bi-modal distribution of pair-wise genetic

distance

Dendrogram of duplicated DNA

samples

Variety identification

Hierarchical clustering dendrogram.

Variety identification

Individual ancestry estimated

Hierarchical clustering dendrogram.

Projection of identified clones on the Ghana map.

Farmer-elicited variety names often do not match specific genotypes

182 different names recorded for the entire sample collection and here only names that occurred > 9 times are represented.

Unique varieties identified using DNA fingerprinting

I II III IV V VI VII VIII X XI IX

Farm

er-

elic

ite

d v

arie

ty n

ame

s

ABENWOHA 2 29 2

AFIA_KOFIE 1 11 1

AMPENKYENE 16

ANKRA 66 8 1 1

BANKYE_KOKOO 27 6 2 1

BOSOMENSIA 2 19 1 11 1 2 1 1

DEBOR 73 7 2

ESIABAYAA 1 22 3 2

KOTEE 2 1 22

TUAKA 1 2 11 1

Variety identification with reduced number of markers

NumberofSNPs

FSTthreshold

0.8

0.85

0.9

0.95

1

1.05

56489

43007

37900

30962

24560

14426

5359

2755

1392

570

324

0 0.10.20.30.40.50.60.70.80.90.95

R^2

FST

Random

Goal: To design smaller set of ancestry informative SNP markers based on African germplasm in cooperation with CIAT.

Further Reading 1. Brookes, A. J. (1999) The essence of SNPs, Gene, 234: 177-86.

2. Cho, R. J., Mindrinos, M., Richards, D. R., Sapolsky, R. J., Anderson, M., Drenkard, E., Dewdney, J., Reuber, T. L.,

Stammers, M., Federspiel, N., Theologis, A., Yang, W. H., Hubbell, E., Au, M., Chung, E. Y., Lashkari, D.,

Lemieux, B., Dean, C., Lipshutz, R. J., Ausubel, F. M., Davis, R. W., and Oefner, P. J. (1999) Genome-Wide

Mapping with Biallelic Markers in Arabidopsis thaliana, Nature Genetics, 23: 203-7.

3. Eglinton, J. K., Langridge, P., and Evans, D. E. (1998) Thermostability Variation in Alleles of Barley Beta-Amylase,

Journal of Cereal Science, 28: 301-309.

4. Griffin, T. J., and Smith, L. M. (2000) Single-Nucleotide Polymorphism Analysis by MALDI-TOF Mass Spectrometry,

Trends in Biotechnology, 18: 77-84.

5. Künzel, G., Korzun, L., Meister, A., and Endo, T. R. (2000) High Resolution Physical Mapping of the Barley

Genome, International Barley Genetics Symposium VIII, I: 293-298.

6. Landegren, U., Nilsson, M., and Kwok, P. Y. (1998) Reading Bits of Genetic Information - Methods for Single

Nucleotide Polymorphism Analysis, Genome Research, 8: 769-776.

7. Nickerson, D. A., Taylor, S. L., Weiss, K. M., Clark, A. G., Hutchinson, R. G., Stengard, J., Salomaa, V., Vartiainen,

E., Boerwinkle, E., and Sing, C. F. (1998) DNA sequence diversity in a 9.7-kb region of the human lipoprotein

lipase gene, Nature Genetics, 19: 233-40.

8. Paris, M., and Carter, M. (2000) Cereal DNA: A Rapid High-Throughput Extraction Method for Marker Assisted

Selection, Plant Molecular Biology Reporter, 18: 357-360.

9. Paris, M., Potter, R., and Jones, M. (2001) Typing Barley Mlo Alleles by Single Nucleotide Polymorphim Analysis

using MALDI-ToF Mass Spectrometry, Plant and Animal Genome IX, P335.

10. Rafalski, A., Ching, A., Bhattramakki, D., Morgante, M., Dolan, M., Register, J. C., Smith, O. S., and Tingey, S.

(2001) SNP Markers in Maize: Discovery and Applications, Plant and Animal Genome IX, W149.

11. Rafalski, A., (2002) Applications of single nucleotide polymorphisms in crop geneticsCurr Opin Plant Biol.

Apr;5(2):94-100

12. Useche, F., Morgante, M., Hanafey, M., Tingey, S., Martins, W., Gao, G. R., and Rafalski, A. (2001) Computer

Detection of Single Nucleotide Polymorphisms (SNPs) in Maize ESTs, Plant and Animal Genome IX, P333.