new tools for measuring - international seed testing ......hence they are ideal for genetic testing....

ISTA Seed Symposium 2010 Cologne - Session 2 – Enrico Noli 1

New tools for measuringgenetic quality in seed

Enrico Noli

ISTA Seed Symposium Session 2 – Aspects of purity: genetic, technical, and

physical


Seedstrategic element for development

• The first step in the food chain

• Seed is the vehicle of genetic innovation

• A steady availability of quality seed is

necessary for the correct functioning of

agricultural production


Physical purity

Freedom from soilborne

diseases and insect damage

Viability/

germination

Vigour

Freedom from mechanical

& heat damage,

pre-harvest sprouting

Uniformity

in size

Seed quality

attributes

Genetic• Variety identity

• Variety purity


Yield increases in Italy 1945-2004

0

2000

4000

6000

8000

10000

12000

1940 1950 1960 1970 1980 1990 2000 2010

Kg

/Ha

Durum wheat

Bread wheat

Maize

Rice

Source Prof. F. Salamini


Source

FAO

Wheat yield increases in developing countries

1950-2004


Plant Breeding

•new genetic diversity

•reassembling existing

diversity

EntomologyStatistics &

bioinformatics

Biotechnology

Plant

pathology

Plant

biochemistry

Plant

physiology Genetics &

genomics

Botany

Agronomy


Cereal production targets

(Tester & Langridge, Science 2010)


Sources of growth in crop production

• expanding the land area

• increase irrigated area

• intensification of cultivation

• boosting yields


• Increase yields/reduce losses

• Improve nutritional values

• Reduce use of pesticides

• Reduce use of fertilizers

• Reduce GHG emissions

• Increase carbon sequestration

• Save land and maintain biodiversity

Contributions of Plant Breeding in

responding to the challenges


Genetic marker

any genetically determined trait

(morfological, biochemical, molecular)

that can distinguish among genotypes

Genetic quality• Variety identity

• Variety purity


On seed On seedlings

In the lab or greenhouse In the field

Traditional approach to variety testing:

Morphological traits


Plant habit Head color

Shape of shoulder

Morphological traits

Test Guidelines for DUS


• A very limited number of discriminatory

traits can be observed directly on seed or

seedlings, a limited number can be assessed

on plants

• In addition for grow-out tests (GOT)

•Long duration and costs of field trials

•Effect of environment on phenotype

•Evaluation sometimes subjective

Limitations of morphological traits


Biochemical markers: electrophoresis of proteins

Isozymes

Phosphoglucoisomerase (PGI)

sunflower seedlings

homozygotes heterozygote

Malate dehydrogenase (MDH)

maize coleoptiles


UPOV Guidelines to DUS testing for maize

Isozymes

Enzyme StructureChromosomic

positionLocus

Malate dehydrogenase MDH Dimeric 8 Mdh1

6L Mdh2

3L Mdh3

1L Mmm

1L Mdh4

5S Mdh5

Isocitrate dehydrogenase IDH Dimeric 8 Idh1

6 Idh2

PGD Dimeric 6 Pgd1

3L Pgd2

Fosfoglucomutasi PGM Monomeric 1L Pgm1

5S Pgm2

Phosfoglucoisomerase PGI Dimeric 1L Pgi1

Acid phosphatase ACP Dimeric 9L Acp1

Diaphorase DIA Monomeric 2 Dia1

Dimeric 1L Dia2

Alcohol dehydrogenase ADH Dimeric 1L Adh1

6-Phoosfogluconate

dehydrogenase


Acid-PAGE of wheat gliadins

Seed storage proteins

source Dr. D. Perry, CGC


Acid pH Basic pHpH = pI

Isoelectrical

Point

Netto

chargeCOOH

NH2OH-

COOHH+

COO-

COO-

COO-

COO-

NH2NH3+

NH3+NH3

+

NH3+

0

+1

+2

+3

-3

-2

-15 10

pH

NET CHARGEISOELECTRIC

POINT

pH

gra

die

nt

ele

ctr

ic f

ield

+

-


Ultra Thin Layer – IsoElectroFocusing - PAGE

source Prof. N. Leist


UTL-IEF PAGE of tomato


F1

selfed


Molecular Markers (MM)

•detect variation directly at DNA level

•mendelian inheritance, often codominant

•highly aboundant and polymorfic

•independent from enviroment and reproducible

•expressed in all developmental stages

•known position in the genome

•sometimes in genes of interest

•automation possibile

hence they are ideal for genetic testing


Wentzl et al. BMC Genomics, 2005

Marker distribution in

the barley genome


MM types and detection techniques

RFLPRestriction Fragment

Length Polymorphism

VNTR

SSLP

STMS

SSRSimple

Sequence

Repeat

EST

SNPSingle

Nucleotide

Polymorphism

CAPSCleaved

Amplified

Polymorphic

Sequences

RAPDRandom Amplified

Polymorphic DNA

AP-PCR

I-SSR

DAF

AFLPAmplified

Fragment

Length

Polymorphism

SAMPL

S-SAP

Single locus Multi - locus

INDELINsertion/

DELetion

polymorphism

Hybridization(Southern Blot )

Amplification

(PCR)


DNA stampo

1° ciclo

21 =

2 copie

2° ciclo

22 =

4 copie

3° ciclo

23 =

8 copie

4° ciclo

24 =

16 copie

gene desiderato

35 ciclo

235 = 34 miliardi di copie

• Very little DNA needed (single seed, leaf punch)

• Fast, simple, non radioactive procedures

• Very high sensitivity and specificity

Techniques based on PCR


INDELINsertion DELetion polymorphism

A B

single locus, biallelic

A

B

ABC D

single locus, multiallelic

C

D

B

A

SSRSimple Sequence Repeats


18

7

18

9

AA

T

C

GB

A

B

Assay “A”

primer “T”

Assay “B”

primer “G”

A BH

A

B

B

Enzimatic digestion

A

CAPCleaved Amplified Polymorphism

SNPSingle Nucleotide Polymorphism


multi-locus (10-15), biallelic multi-locus (50-100), biallelic


n. loci

n.

allele

s

RFLPsinglecopy

SSR

RAPD AFLP

RFLPVNTR

SNP

INDEL

CAPS

Marker informativeness


A. Breeding new varieties

• Characterization and use of germplasm (pre-breeding)

• Selection process (Marker Assisted Selection)

B. Variety registration/protection of PBRs

• DUS assessment

• Identification of EDV

C. Seed production and control

• Variety identity and purity

MM applications in breeding and seed production


•Profiles of 2 SSR loci flanking a

resistance gene on chromosome 5A

•P1 = resistant parent, P2 =

susceptible parent, 12 F2 plants

•At both loci the A allele is

associated to resistance

•Plants 2 and 3 will be selected

because homozygous for the

―resistant‖ allele at both loci

F2

P1

P2

M’

1 2 4 6 83 5 7 9 10 11 12

A

B

M’’

A

B

R

r

M’A M’’A

M’B M’’B

MAS for resistance to Fusarium head blight in wheat

A. Breeding new varieties

P1

P2


The necessity of recouping the

investments for variety

development has lead to a legal

framework for the protection of

Plant Breeder’s Rights against

misappropriations

B. Variety registration/protection of PBRs

- DUS assessment

- Identification of EDV


Group of Biochemical and Molecular

Techniques and DNA profiling — BMT

DUS assessment

1. As predictors of descriptors (MM tightly linked or on

genes)

2. For the management of reference collections

BMT options for the use of MM for DUS

(varieties to be compared in field

trials with the candidate variety

should be the most similar at the

molecular level)

3. As a completely new system


Identification of Essentially Derived Varieties (EDV)

original

essentially

derived (EDV)

genetic

distance

independent

new variety

threshold value

determined with MM

Obtained from an original variety through

genetic engineering, backcrossing, selection of mutants


1. AFLP analysis of a defined set of independent varieties

2. Calculate genetic similarity between varieties

0.00 1.00

threshold

3. Define the threshold for EDV as the 95

percentile of the distribution of similarities

4. Estimate with AFLP the genetic similarity between new and old vaiety

5. If the threshold value of similarity is exceeded, the breeder of the new

variety will have the burden to demonstrate that the variety is

independent

Example protocol for EDV

3.2.

1.


C. Seed production - Variety ID

36 most popular Italian durum wheat varieties


Variety ID

Xb

arc

36

0

Cfa

21

23

Wm

s 4

94

Xb

arc

3

Wm

s1

55

wm

s4

13

Achille 340 278 227 210 147 113

Anco_Marzio 340 262 227 210 154 107

Ariosto 340 278 227 210 147 113

Aureo 331 262 232 215 164 113

Avispa 340 264 227 210 147 107

Biensur 900 284 227 210 147 113

Catervo 340 255 225 210 147 107

Ciccio 338 278 232 210 146 107

Claudio 338 271 240 210 147 107

Colosseo 340 273 230 210 147 120

Creso 340 278 230 210 147 113

Dario 340 278 232 210 146 113

Duilio 340 278 227 210 147 113

Dylan 340 278 230 210 147 107

Grecale 900 284 227 210 147 107

Imothep 900 278 227 210 147 107

Iride 340 262 227 210 147 107

Isildur 340 264 230 210 147 113

K26 338 258 230 212 162 107

Latinur 340 282 227 210 147 113

Levante 327 257 232 210 160 107

Liberdur 340 278 227 210 147 113

Maestrale 320 278 227 202 147 113

Matt 340 284 225 210 147 107

Meridiano 340 264 227 210 147 107

Miradux 340 284 227 210 147 107

Neolatino 340 264 230 210 147 113

Normanno 338 262 227 210 147 107

Orobel 338 273 227 210 147 113

Database development (durum wheat)

107

147

210

227

262

340

Allele

(bp)

Xgwm413

Xgwm155

Xbarc3

Xwmc494

XCfa2123

Xbarc360

loci

Unknown sample


Variety ID

Xb

arc

36

0

Cfa

21

23

Wm

s 4

94

Xb

arc

3

Wm

s1

55

wm

s4

13

Anco_Marzio 340 262 227 210 154 107

Iride 340 262 227 210 147 107

Database development (durum wheat)

107

147

210

227

262

340

Allele

(bp)

Xgwm413

Xgwm155

Xbarc3

Xwmc494

XCfa2123

Xbarc360

loci

Unknown sample


Variety ID

Xb

arc

36

0

Cfa

21

23

Wm

s 4

94

Xb

arc

3

Wm

s1

55

wm

s4

13

Iride 340 262 227 210 147 107

107

147

210

227

262

340

Allele

(bp)

Xgwm413

Xgwm155

Xbarc3

Xwmc494

XCfa2123

Xbarc360

loci

Unknown sample


Variety ID

Traceability: monovarietal pasta

Xbarc360

XCfa2123

Xbarc3

Xwmc494

Xgwm155

Xgwm413

Se

ed

-SIS

Se

ed

-P

SB

Pa

sta

-XX

X

364

350

300

255

204

200

145

100

Results

•3/6 loci show difference between

pasta and seed sources

•Pasta maker did not use the

declared variety


These plants lack the band chracteristic of male parent,

so they must derive from selfing of the female

Assessment of genetic purity of F1 tomato seed

RAPD

SSR

C. Seed production – Varietal purity



Genetic homogeneity in sunflower inbreds

by SSR multiplex

255

300

350364

400

204

460

145

200

line1 line2 line5line3 line7line6line4 line8 line9 line12line10line11



sv

me

17

5

17

7

17

8

18

01

81

18

7

18

9

19

3

19

4

19

5

19

6

19

7

19

9

20

0

17

5

17

7

17

8

18

01

81

18

7

18

9

19

3

19

4

19

5

19

6

19

7

19

9

18

5

18

6

18

7

18

8

18

9

19

0

19

1

19

2

19

3

19

4

19

5

19

6

sv

me

20

0

18

5

18

6

18

7

18

8

18

9

19

0

19

1

19

2

19

3

19

4

19

5

19

6

Wild type assay

(non-tolerant plants)

Mutant assay

(tolerant plants)

Specified trait: herbicide tolerance by SNP


Tests for quantification of GM in seeds

qualitative

assays

quantitative

assays

assessment of

trait purity in GM

seed lots

bulk analysis of the

whole sample

semi-quantitative assays

analysis of bulks of

seeds (subsamples)

analysis of individual

seeds in the sample

assessment of adventitious

presence (AP) of GM seeds in

conventional seed lots”


GM trait purity (%)

n° GM seed * 100

n° germinated seeds

GM contamination (%)

+ herbicide

non-GM

GM

ELISA

rt qPCR

end-point PCR

P P P P P P P P P PNN

immunoassay

qualitative

assays

quantitative

assays

semi-quantitative assays


High trait purity Low AP level

100 50 25 12.5 6.25 3.13 1.56 0.78

GM seed Conventional seed

GMO %

0

0

0

1

1

1

1

1

2

2

2

2

2

12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

100%

50.0%

25.0%

12.5%

6.25%

3.13%

1.56%

0.78%

Amplification cycle

Flu

ore

sce

nce

Threshold


PCR walking and

sequencing

Primer and

TaqMan probe

design

GM allele

plant genomeGM insert

5’ 3’unknownknown

known

wt allele

plant genomeplant genome

5’ 3’unknown known

known


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 RR ntc21 22 23 24 25 26 27 28 29 30 m

w

mw

Amplification of wt allele in 30 conventional soybean varieties

• The wt sequence is highly conserved

• Low risk of false negatives

• The assay seems to allow the detection of any wt contaminant

Amplification of wt allele in 20 Roundup Ready soybean varieties

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 wt ntc m

wmw

• The wt sequence is present as a single copy in the genome

• Low risk of false positives

• The assay seems suitable to test purity of any RR soybean variety

wt

wt


rtqPCR for wt allele vs current methods

No toxic chemicals needed

Objectivity of test result – no doubts

Compared to bioassays

Shorter time of analysis

A single analysis on a bulk is sufficient for a quantitative result

Sample size can be significantly increased for higher representativity

Compared to all other methods

Cost of analysis basically unaffected by sample size (whereas it

depends on # seeds for current methods)


Conclusions

MM are very efficient tools for VarID and purity

There is a great aboundance of sequences that can be

used, both ―anonimous‖ and of known function

Markers in tight linkage or within genes controlling

traits of interest are of great value when testing for

genetic quality

The same markers and analytical systems can be used

for variety development, management of seed

production and qualiy control, and to ensure

traceability downstream in the food chain

This area can provide opportunities and challenges for

seed testing laboratories


Aknowledgement

Elena Battistini

Emanuela Casarini

Silvia Scacchi

Maria Teriaca

Thank for your attention!

new tools for measuring - international seed testing ......hence they are ideal for genetic testing....

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