New tools for measuring genetic quality in seed

Transcription

1 ISTA Seed Symposium Session 2 Aspects of purity: genetic, technical, and physical New tools for measuring genetic quality in seed Enrico Noli ISTA Seed Symposium 2010 Cologne - Session 2 Enrico Noli 1

2 Seed strategic 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 ISTA Seed Symposium 2010 Cologne - Session 2 Enrico Noli 2

3 Freedom from soilborne diseases and insect damage Physical purity Freedom from mechanical & heat damage, pre-harvest sprouting Viability/ germination Seed quality attributes Uniformity in size Vigour Genetic Variety identity Variety purity ISTA Seed Symposium 2010 Cologne - Session 2 Enrico Noli 3

4 Kg/Ha Yield increases in Italy Durum wheat Bread wheat Maize Rice Source Prof. F. Salamini ISTA Seed Symposium 2010 Cologne - Session 2 Enrico Noli 4

5 Wheat yield increases in developing countries Source FAO ISTA Seed Symposium 2010 Cologne - Session 2 Enrico Noli 5

6 Entomology Statistics & bioinformatics Plant pathology Plant biochemistry Plant Breeding new genetic diversity reassembling existing diversity Biotechnology Agronomy Plant physiology Genetics & genomics Botany ISTA Seed Symposium 2010 Cologne - Session 2 Enrico Noli 6

7 Cereal production targets (Tester & Langridge, Science 2010) ISTA Seed Symposium 2010 Cologne - Session 2 Enrico Noli 7

8 Sources of growth in crop production expanding the land area increase irrigated area intensification of cultivation boosting yields ISTA Seed Symposium 2010 Cologne - Session 2 Enrico Noli 8

9 Contributions of Plant Breeding in responding to the challenges 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 ISTA Seed Symposium 2010 Cologne - Session 2 Enrico Noli 9

10 Genetic quality Variety identity Variety purity Genetic marker any genetically determined trait (morfological, biochemical, molecular) that can distinguish among genotypes ISTA Seed Symposium 2010 Cologne - Session 2 Enrico Noli 10

11 Traditional approach to variety testing: Morphological traits On seed On seedlings In the lab or greenhouse In the field ISTA Seed Symposium 2010 Cologne - Session 2 Enrico Noli 11

12 Morphological traits Plant habit Head color Shape of shoulder Test Guidelines for DUS ISTA Seed Symposium 2010 Cologne - Session 2 Enrico Noli 12

13 Limitations of morphological traits 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 ISTA Seed Symposium 2010 Cologne - Session 2 Enrico Noli 13

14 Biochemical markers: electrophoresis of proteins Isozymes Malate dehydrogenase (MDH) maize coleoptiles homozygotes heterozygote Phosphoglucoisomerase (PGI) sunflower seedlings ISTA Seed Symposium 2010 Cologne - Session 2 Enrico Noli 14

15 Isozymes UPOV Guidelines to DUS testing for maize Enzyme Structure Chromosomic position Locus Malate dehydrogenase MDH Dimeric 8 Mdh1 6L Mdh2 3L Mdh3 1L Mmm 1L Mdh4 5S Mdh5 Isocitrate dehydrogenase IDH Dimeric 8 Idh1 6 Idh2 6-Phoosfogluconate PGD Dimeric 6 Pgd1 dehydrogenase 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 ISTA Seed Symposium 2010 Cologne - Session 2 Enrico Noli 15

16 Seed storage proteins Acid-PAGE of wheat gliadins source Dr. D. Perry, CGC ISTA Seed Symposium 2010 Cologne - Session 2 Enrico Noli 16

17 ph gradient electric field Seed storage proteins Ultra Thin Layer IsoElectroFocusing - PAGE Netto charge COOH NH 3 + COO - Isoelectrical Point NH + OH - 3 COO - NH 2 COOH H+ COO - COO - NH 3 + NH 3 + NH 2 Acid ph ph = pi Basic ph NET CHARGE ISOELECTRIC POINT ph + - source Prof. N. Leist ISTA Seed Symposium 2010 Cologne - Session 2 Enrico Noli 17

18 Seed storage proteins UTL-IEF PAGE of tomato F1 selfed ISTA Seed Symposium 2010 Cologne - Session 2 Enrico Noli 18

19 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 ISTA Seed Symposium 2010 Cologne - Session 2 Enrico Noli 19

20 Marker distribution in the barley genome Wentzl et al. BMC Genomics, 2005 ISTA Seed Symposium 2010 Cologne - Session 2 Enrico Noli 20

21 MM types and detection techniques Hybridization (Southern Blot ) Amplification (PCR) RFLP Restriction Fragment Length Polymorphism VNTR SSLP STMS SSR Simple Sequence Repeat CAPS Cleaved Amplified Polymorphic Sequences INDEL INsertion/ DELetion polymorphism EST SNP Single Nucleotide Polymorphism RAPD Random Amplified Polymorphic DNA AP-PCR I-SSR DAF AFLP Amplified Fragment Length Polymorphism SAMPL S-SAP Single locus Multi - locus ISTA Seed Symposium 2010 Cologne - Session 2 Enrico Noli 21

22 Techniques based on PCR gene desiderato 3 ciclo 4 ciclo 2 ciclo DNA stampo 1 ciclo 35 ciclo 2 1 = 2 copie 2 2 = 4 copie 2 3 = 8 copie 2 4 = 16 copie 2 35 = 34 miliardi di copie Very little DNA needed (single seed, leaf punch) Fast, simple, non radioactive procedures Very high sensitivity and specificity ISTA Seed Symposium 2010 Cologne - Session 2 Enrico Noli 22

23 INDEL INsertion DELetion polymorphism SSR Simple Sequence Repeats A B C D B A single locus, biallelic single locus, multiallelic A B C D B A ISTA Seed Symposium 2010 Cologne - Session 2 Enrico Noli 23

24 SNP Single Nucleotide Polymorphism CAP Cleaved Amplified Polymorphism A Assay A primer T A H B B Assay B primer G A A B T A G C B A B Enzimatic digestion ISTA Seed Symposium 2010 Cologne - Session 2 Enrico Noli 24

25 multi-locus (10-15), biallelic multi-locus (50-100), biallelic ISTA Seed Symposium 2010 Cologne - Session 2 Enrico Noli 25

26 n. alleles Marker informativeness SSR RFLP VNTR SNP INDEL CAPS RFLP single copy RAPD AFLP n. loci ISTA Seed Symposium 2010 Cologne - Session 2 Enrico Noli 26

27 MM applications in breeding and seed production 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 ISTA Seed Symposium 2010 Cologne - Session 2 Enrico Noli 27

28 P1 P2 M M A B A B A. Breeding new varieties MAS for resistance to Fusarium head blight in wheat F P1 P2 M A M B R 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 r M A M B ISTA Seed Symposium 2010 Cologne - Session 2 Enrico Noli 28

29 B. Variety registration/protection of PBRs - DUS assessment - Identification of EDV 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 ISTA Seed Symposium 2010 Cologne - Session 2 Enrico Noli 29

30 DUS assessment Group of Biochemical and Molecular Techniques and DNA profiling BMT BMT options for the use of MM for DUS 1. As predictors of descriptors (MM tightly linked or on genes) 2. For the management of reference collections (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 ISTA Seed Symposium 2010 Cologne - Session 2 Enrico Noli 30

31 Identification of Essentially Derived Varieties (EDV) Obtained from an original variety through genetic engineering, backcrossing, selection of mutants new variety original essentially derived (EDV) independent threshold value determined with MM genetic distance ISTA Seed Symposium 2010 Cologne - Session 2 Enrico Noli 31

32 1. Example protocol for EDV 1. AFLP analysis of a defined set of independent varieties 2. Calculate genetic similarity between varieties Define the threshold for EDV as the 95 percentile of the distribution of similarities threshold 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 ISTA Seed Symposium 2010 Cologne - Session 2 Enrico Noli 32

33 C. Seed production - Variety ID 36 most popular Italian durum wheat varieties ISTA Seed Symposium 2010 Cologne - Session 2 Enrico Noli 33

34 Xbarc360 Cfa2123 Wms 494 Xbarc3 Wms155 wms413 Variety ID Database development (durum wheat) Achille Anco_Marzio Ariosto Aureo Avispa Biensur Catervo Ciccio Claudio Colosseo Creso Dario Duilio Dylan Grecale Imothep Iride Isildur Unknown sample loci Xbarc360 XCfa2123 Xwmc494 Xbarc3 Xgwm155 Xgwm413 Allele (bp) ISTA Seed Symposium 2010 Cologne - Session 2 Enrico Noli 34

35 Xbarc360 Cfa2123 Wms 494 Xbarc3 Wms155 wms413 Variety ID Database development (durum wheat) Anco_Marzio Iride Unknown sample loci Xbarc360 Allele (bp) 340 XCfa2123 Xwmc494 Xbarc Xgwm155 Xgwm ISTA Seed Symposium 2010 Cologne - Session 2 Enrico Noli 35

36 Xbarc360 Cfa2123 Wms 494 Xbarc3 Wms155 wms413 Variety ID Unknown sample loci Allele (bp) Iride Xbarc XCfa2123 Xwmc494 Xbarc Xgwm155 Xgwm ISTA Seed Symposium 2010 Cologne - Session 2 Enrico Noli 36

37 Seed-SIS Pasta-XXX Seed -PSB Variety ID Traceability: monovarietal pasta Xbarc XCfa Xbarc3 Xwmc494 Xgwm155 Xgwm Results 3/6 loci show difference between pasta and seed sources Pasta maker did not use the declared variety ISTA Seed Symposium 2010 Cologne - Session 2 Enrico Noli 37

38 C. Seed production Varietal purity Assessment of genetic purity of F1 tomato seed RAPD SSR These plants lack the band chracteristic of male parent, so they must derive from selfing of the female ISTA Seed Symposium 2010 Cologne - Session 2 Enrico Noli 38

39 C. Seed production Varietal purity Genetic homogeneity in sunflower inbreds by SSR multiplex line1 line2 line3 line4 line5 line6 line7 line8 line9 line10line11 line ISTA Seed Symposium 2010 Cologne - Session 2 Enrico Noli 39

40 sv me sv me C. Seed production Varietal purity Specified trait: herbicide tolerance by SNP Wild type assay (non-tolerant plants) Mutant assay (tolerant plants) ISTA Seed Symposium 2010 Cologne - Session 2 Enrico Noli 40

41 Tests for quantification of GM in seeds qualitative assays semi-quantitative assays quantitative assays analysis of individual seeds in the sample analysis of bulks of seeds (subsamples) bulk analysis of the whole sample assessment of trait purity in GM seed lots assessment of adventitious presence (AP) of GM seeds in conventional seed lots ISTA Seed Symposium 2010 Cologne - Session 2 Enrico Noli 41

42 qualitative assays semi-quantitative assays quantitative assays non-gm + herbicide GM end-point PCR ELISA n GM seed * 100 n germinated seeds N N P P P P P P P P P P immunoassay rt qpcr GM trait purity (%) GM contamination (%) ISTA Seed Symposium 2010 Cologne - Session 2 Enrico Noli 42

43 Fluorescence GM seed Conventional seed GMO % High trait purity Low AP level % 50.0% 25.0% 12.5% 6.25% 3.13% 1.56% 0.78% 0 0 Threshold Amplification cycle ISTA Seed Symposium 2010 Cologne - Session 2 Enrico Noli 43

44 GM allele wt allele 5 known unknown 3 GM insert plant genome 5 unknown known 3 plant genome plant genome PCR walking and sequencing known Primer and TaqMan probe design known ISTA Seed Symposium 2010 Cologne - Session 2 Enrico Noli 44

45 mw Amplification of wt allele in 30 conventional soybean varieties RR ntc m w wt 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 mw wt ntc m w wt 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 ISTA Seed Symposium 2010 Cologne - Session 2 Enrico Noli 45

46 ISTA Seed Symposium 2010 Cologne - Session 2 Enrico Noli 46

47 rtqpcr for wt allele vs current methods Compared to bioassays Objectivity of test result no doubts No toxic chemicals needed Shorter time of analysis Compared to all other methods A single analysis on a bulk is sufficient for a quantitative result Sample size can be significantly increased for higher representativity Cost of analysis basically unaffected by sample size (whereas it depends on # seeds for current methods) ISTA Seed Symposium 2010 Cologne - Session 2 Enrico Noli 47

48 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 ISTA Seed Symposium 2010 Cologne - Session 2 Enrico Noli 48

49 Aknowledgement Elena Battistini Emanuela Casarini Silvia Scacchi Maria Teriaca Thank for your attention! ISTA Seed Symposium 2010 Cologne - Session 2 Enrico Noli 49