1 Genotypic Analysis: Methods, Considerations, and Examples Biology, Ecology, & Genetics of Forest Diseases March 19, 2009

2 Study the genetic structure of a population in an area Number of different genotypes Determine gene flow between two population Determine if there is an ongoing invasion Duration of infestation

3 Some Considerations in Choosing a Genotyping Method Level of taxonomic resolution desired (Populations? Species? Phyla?) Level of genotypic resolution desired –Dominant vs. codominant markers –Fine (e.g., nucleotide-level) data vs. coarse (e.g., fragment size) genomic scale Previous sequence knowledge Cost and labor constraints

4 Genetic Markers SNPsSingle Nucleotide Polymorphisms substitution of a nucleotide 4 alleles: Adenine, Guanine, Cysteine, Thymine Insertion/deletion of a nucleotide 2 alleles: presence or absence of the nucleotide Approximately every 200 – 300 bp Different degrees of variability Microsatellites variation in number of short tandem repeats Unknown number of alleles High variability

5 Choice of genetic marker (a) Comparison of individuals of the same species but isolated requires markers with low level of variability No microsatellites SNPs in genes necessary for the survival of the cell ATPase (cellular energy) Cyt b (cytochrome b) Cox1 (cytochrome c oxidase subunit 1)

6 Choice of genetic marker (b) Comparison of individuals closely related requires markers with high level of variability Microsatellites SNPs in non-coding regions of genes Anonymous SNPs in the genome

7 PCR amplification (a) PCR amplification requires:  DNA template  2 oligonucleotides - Primers  Nucleotides dATP, dCTP, dGTP, dTTP  Taq polymerase

8 PCR reaction (b) 1.Double strand denaturation 2.Annealing of the primers 3.Elongation 5’ 3’

9 Restriction Enzymes Found in bacteria Cut DNA within the molecule (endonuclease) Cut at sequences that are specific for each enzyme (restriction sites) Leave either blunt or sticky ends, depending upon the specific enzyme Tobin & Dusheck, Asking About Life, 2nd ed. Copyright 2001, Harcourt, Inc.

10 Microsatellites Short tandem repeats ACT DNA Microsatellites are located in non-coding regions

11 Fluorescent genotyping of microsatellites 1.PCR amplification using 1 primer fluorescently labeled 2.PCR amplification product mixed with a size marker 3.PCR fragments separated by capillary electrophoresis ACT 5’

12  Size of the amplification product is variable and corresponds to the length of the flanking sequences plus a multiple of the size of the repeat  Co-dominant: homozygote for allele 1 homozygote for allele 2 heterozygote

13 Tetra repeat: allele bp

14 Tetra repeat: allele bp

15 Sequencing reaction (a) Sequencing reaction requires:  PCR amplification product as template  1 oligonucleotide - Primer  Nucleotides dATP, dCTP, dGTP, dTTP  Taq polymerase  Modified nucleotides ddATP, ddCTP, ddGTP, ddTTP –ddNTPs are incorporated into the polynucleotide chain and block further elongation –ddNTPs are fluorescently labeled, each with a different fluorocrome

16 Sequencing reaction (b) 1.Annealing 2.Elongation 3.Incorporation of ddNTP and stop of the elongation ddATP FAM ddCTP HEX 5’

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18 Alignment of the 2 sequences obtained using the Forward and the Reverse primers on the same PCR amplification product

19 Alignment of several sequences showing a T/C substitution (homozygote)

20 PCR-RFLP Restriction Fragment Length Polymorphism Restriction enzymes cut the DNA at specific sequences DNA fragment containing a restriction sequence (EcoRI) AGGTGAATCCAAAATTTT DNA fragment after restriction digestion AGGTG AATTCAAATTT

21 Scoring PCR-RFLP  PCR amplification of the region containing the restriction sites  Electrophoresis to identify presence or absence of bands Size marker Sample 1 Sample 2

22 PCR-RFLP Fluorescent electrophoresis

23 P. ramorum CoxI-PCR-RFLP PCR amplification of a 972 bp portion of the CoxI gene Restriction digestion with Apo I EU isolates (mating type A1) have a C at position 377 of the amplicon Apo I cuts US isolates (mating type A2) have a T at position 377 of the amplicon Apo I does not cut

24 PCR-SSCP Single Strand Conformation Polymorphisms Denatured DNA (single strand) can be differentiate using electrophoresis on the basis of a single nucleotide difference  PCR amplification of region containing the polymorphism  Denaturation  Gel electrophoresis

25 PCR amplification of a selected gene, with one primer labeled with a fluorophore. Digestion of DNA with a restriction enzyme; number and length of the resulting fragments is determined by the presence/absence of appropriate restriction sites (i.e., depends upon the underlying DNA sequence Because the fluorophore is bound to the 5’ end of the PCR product, only the fragment that occurs 5’ to the restriction site will appear when run on an automated DNA sequencer Size of the fragment may be specific to a certain genotype (though resolution is limited!) T-RFLP Terminal Restriction Fragment Length Polymorphisms

26 T-RFLP Analysis I: Hierarchical Clustering Grouping by overall similarity (distance) calculated between plots or communities -- e.g., Jaccard’s index: J=M/(M+N), where M = #matches and N= #mismatches; followed by clustering (e.g., UPGMA) Figure: Plots clustered by bacterial community composition. Groupings do not correspond to carbon dioxide enrichment treatment (Osmundson, Naeem et al., in prep.)

27 T-RFLP Analysis II: MRPP & Indicator Species Analysis Multiresponse permutation procedure (MRPP): Do a priori groups (in this example, based on carbon dioxide treatment) differ significantly in their biotic (in this example, microbial) communities? Indicator Species Analysis: Are there species that discriminate between groups? (Osmundson, Naeem et al., in prep)

28 T-RFLP Analysis III: NMS (Nonmetric Multidimensional Scaling) Ordination based on community presence/absence matrix

29 Random Genomic Markers DNA sequence of suitable SNPs is not available Relatively inexpensive Scan the entire genome producing information on several variations in the same reaction  RAPD Random Amplification of Polymorphic DNA  AFLP Amplified Fragment Length Polymorphism

30 RAPD Random Amplification of Polymorphic DNA Amplification of genomic DNA included between 2 identical short sequences (random) Genomic DNA is amplified with 1 pair of identical (complementary) primers (generally 10 bp and GC rich) example: 5’ AATCGGTACA 3’ and 5’ TGTACCGATT 3’ Amplification using a low annealing temperature (increased amplification for sequences not exactly complementary to the primer sequence) The primers amplify or not depending on the presence or absence of the short sequence used to design the primers 3’ 5’

31 Scoring RAPD Presence (1) or absence (0) of amplification product = Dominant marker  Mismatches between primer and template might also result in decreased amount of PCR product Nucleotide substitution at 3’ end of the primer  no annealing = no amplification Nucleotide substitution at 5’ end of the primer  < annealing = < amplification

32 AFLP Amplified Fragment Length Polymorphisms (Vos et al., 1995)  Genomic DNA digested with 2 restriction enzymes: –EcoRI (6 bp restriction site) cuts infrequently –MseI(4 bp restriction site) cuts frequently GAATTC CTTAAG TTAA AATT

33  Fragments of DNA resulting from restriction digestion are ligated with end-specific adaptors (a different one for each enzyme) to create a new PCR priming site  Pre selective PCR amplification is done using primers complementary to the adaptor + 1 bp (chosen by the user) NNN N

34  Selective amplification using primers complementary to the adaptor (+1 bp) + 2 bp NNN

35 AFLP genotyping PCR amplification using primers corresponding to the new sequence If there are 2 new priming sites within 400 – 1600 bp there is amplification The result is: Presence or absence of amplification 1 or 0 Dominant marker: does not distinguish between heterozygote and homozygote Due mostly to SNPs but also to deletions/insertions

36 AFLP OVERVIEW (VOS ET AL., 1995)

37 AFLP Fluorescent electrophoresis

38 Analysis  Similarity (cluster analysis) NJ (Neighbor Joining) UPGMA (Un weighted Pair Group Method with Arithmetic mean) AMOVA (Analysis of Molecular Variance)  Maximum likelihood  Parsimony  Bayesian

39 Statistical support  Re-sampling (~ times) Bootstrap analysis The original loci are randomly re-sampled with replacement Jacknife analysis From the original data 1 locus is randomly removed

40 Spatial autocorrelation Moran’s I (coefficient of departure from spatial randomness) correlates with distance up to Distribution of genotypes (6 microsatellite markers) in different populations of P.ramorum in California

41 Spatial autocorrelation Geographical distance (m) Moran’s I 0 Within approx. 100 meters the genetic structure correlates with the geographical distance

42 Example 1: Origins of the Sudden Oak Death Epidemic in California (Mascheretti et al., Molecular Ecology (2008) 17: ) Photo: UC Davis Photo: Photo: Northeast Plant Diagnostic Network

43 NJ tree of P. ramorum populations in California SC-1 MA-4 NURSERY SC-3 MA-3 SO-1 SO-2 MA-5 SC-2 MO-1 MO-2 MA-2 MA-1 HU-1 HU-2

44 Phytophthora ramorum (Oomycete) –causal agent of Sudden Oak Death (SOD) first reported in California in 1994 –SOD affects tanoak (Lithocarpus densiflora), coast live oak (Quercus agrifolia), Californian black oak (Quercus kelloggii), and Canyon live oak (Quercus chrysolepis) –P.ramorum also cause a disease characterized mostly by leaf blight and/or branch dieback in over 100 species of both wild and ornamental plants, including California bay laurel (Umbellularia cailfornica), California redwood (Sequoia sempervirens), Camellia and Rhododrendron species Example: microsatellites genotyping of P. ramorum isolates Collection of infected bay leaves from several forests in Sonoma, Monterey, Marin, Napa, Alameda, San Mateo

45 Microsatellites (I) mating type A1 (EU) and mating type A2 (US) A2 (US)A1 (EU) Locus 29325/ - 325/337 -/337 Locus 33315/337325/337 Locus 65234/ / /222

46 Microsatellites (II) mating type A2 (US) MS39a(GA) bp MS39b(GA) 4 (GATA) , 242, 246, 250, 254 bp MS43a(CAGA) , 329, 349, 353, 357, 361, 365, 369, 373, 377, 381 bp MS43b(CAGA) 75 (…)(CAGA) , 420, 466, 470, 474, 476, 478, 482, 486, 490, 494, 498 bp MS45(TCCG) , 183, 187 bp MS18(AC) , 220, 222, 254, 264, 272, 274, 276, 278, 282 bp MS64(CT) , 374, 376, 378 bp

47 Ind.MS39aMS39bMS43aMS43bMS45MS18MS64 Mating type A A A A A A A A A A A A A A A A A A null A null A2

48 MS18(AC) bp (AC) bp (AC) bp MS43a(CAGA) bp MS43a(CAGA) bp MS43a(CAGA) bp ( ) ( ) ( ) ( ) (39-38) (40-38) (71-70) (72-70) ACACACACACACACACAC AMOVA Analysis of Molecular Variance

49 Ind.MS39aMS39bMS43aMS43bMS45MS18MS64 Mating type A A A A A A A A A A A A A A A A A A null18-29 A null18-29 A2

50 China Camp State Park (Marin)n=24 Bean Creek Forest (Santa Cruz)n=24 Nurseriesn=14

51 Example 2: Distinguishing taxa in the Pleurotus eryngii (King Oyster Mushroom) complex using AFLPs (Urbanelli et al., Appl. Microbiol. Biotechnol. (2007) 74: ) Photo: The New York Times Photo: Wikimedia Commons

52 Distinguishing taxa in the Pleurotus eryngii (King Oyster Mushroom) complex using AFLPs Goal: to determine, using multilocus genotypes, whether the distinction between Pleurotus eryngii, P. ferulae, and P. eryngii var. nebrodensis is supported by genetic data 90 populations sampled 94 AFLP loci scored P. ferulae (

53 Sample AFLP Gel

54 AFLP Data Map from Urbanelli et al. (2007)

55 AFLP Data Map with UPGMA dendogram from Urbanelli et al. (2007)