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Structural genomics in Fragaria x ananassa Denise Cristina Manfrim Tombolato Challenges during strawberry production Diseases - Florida: anthracnose Pests Challenges for Florida strawberry production Diseases Pests Phase-out of methylbromide Market competition/consolidation

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Structural genomics in Fragaria x ananassa Denise Cristina Manfrim Tombolato

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Structural genomics in fragaria x ananassa denise cristina manfrim tombolato l.jpg

Structural genomics in Fragaria x ananassaDenise Cristina Manfrim Tombolato


Challenges during strawberry production l.jpg

Challenges during strawberry production

  • Diseases - Florida: anthracnose

  • Pests


Challenges for florida strawberry production l.jpg

Challenges for Florida strawberry production

  • Diseases

  • Pests

  • Phase-out of methylbromide

  • Market competition/consolidation


One remedy plant improvement l.jpg

One Remedy = Plant improvement

  • Traditional strawberry breeding

    • Desirable fruit quality (appearance, size, firmness, flavor)

    • Open-plant habit (for efficient harvesting and pesticide application)

    • Disease/pest resistance

    • Precocious, prolific cultivars

  • Molecular approaches

    • Structural genomics

      • Marker-assisted selection

    • Functional genomics

      • Analysis of gene function

      • Transgenic plants


One remedy plant improvement5 l.jpg

One Remedy = Plant improvement

  • Traditional strawberry breeding

    • Desirable fruit quality (appearance, size, firmness, flavor)

    • Open-plant habit (for efficient harvesting and pesticide application)

    • Disease/pest resistance

    • Precocious, prolific cultivars

  • Molecular approaches

    • Structural genomics MOLECULAR MARKERS

      • Marker-assisted selection

    • Functional genomics

      • Analysis of gene function

      • Transgenic plants


Uses of molecular markers l.jpg

Uses of Molecular Markers

  • Genetic characterization of plants and animals

    • Conservation of germplasm

    • Insights on evolution

    • Proprietary issues

  • Marker-assisted selection

  • Linkage maps

    • Map-based positional cloning

    • Comparative mapping

      • Detection of chromosome rearrangements between related taxa


Molecular markers in strawberry l.jpg

Molecular Markers in Strawberry

  • Isozymes

    • Cultivar identification

      • Arulsekar et al. 1981

      • Bringhurst et al. 1981

      • Nehra et al. 1991

      • Bell and Simpson, 1994

    • Linkage mapping

      • fruit color & shikimate dehydrogenase isozyme locusWilliamson et al. 1995

      • runnering & Pgi-2 isozyme locusYu & Davis1995

  • Intron length polymorphism

    • Primers specific for genes in the anthocyanin biosynthesis pathwayDeng and Davis 2001


Molecular markers in strawberry8 l.jpg

Molecular Markers in Strawberry

  • Randomly Amplified Polymorphim DNA (RAPD)

    • Cultivar identification

      • Gidoni et al. 1994

      • Hancock et al. 1994

      • Levi et al. 1994

      • Parent & Page, 1995

      • Graham et al. 1996

      • Landry et al., 1997

      • Harrison et al. 1997

      • Degani et al. 1998

      • Degani et al. 2001

    • Linkage mapping in the diploid F. vesca

      • Davis and Yu, 1997

    • Marker-assisted selection

      • Rpf1 = resistance gene to race 1 of Phytophthora fragariae Haymes et al. 1997

      • Rpf2 = resistance gene to race 2, Van de Weg 1997


Molecular markers in strawberry9 l.jpg

Molecular Markers in Strawberry

  • Inter Simple Sequence Repeat (ISSR)

    • Cultivar identification Arnau et al. 2003

    • Marker-assisted selection to seasonal flowering

      • SCAR markers, derived from ISSR Albani et al. 2004

  • Sequence-Characterized Amplified Region (SCAR)

    • Davis et al. 1995

    • Haymes et al. 2000

    • Albani et al. 2004

  • AFLP

    • Cultivar identification Degani et al. 2001

    • Linkage mapping in F. x ananassaLerceteau-Kohler et al. 2003


Molecular markers in strawberry10 l.jpg

Molecular Markers in Strawberry

  • Simple Sequence repeat (SSR)

    • Diploids

      • F. viridis Sargent et al. 2003

      • F. vesca James et al. 2003

      • F. vesca Hadonou et al. 2004

      • F. vesca x F. nubicola map Sargent et al. 2004

      • F. vesca , transferable to other Fragaria Hadonou et al. 2004

      • F. vesca + 5 other diploids Monfort et al. 2005

    • Octoploids

      • F. x ananassa Nourse et al. 2002

      • F. virginiana Ashley et al. 2003

      • F. x ananassa Monfort 2005


Published strawberry maps l.jpg

Published Strawberry Maps

  • Diploid F. vesca

    • RAPD Davis and Yu, 1997

    • SSR Sargent et al. 2004

  • Octoploid

    • AFLP Lerceteau-Köhler et al., 2003


Published strawberry maps12 l.jpg

Published Strawberry Maps

  • Diploid F. vesca

    • RAPD Davis and Yu, 1997

    • SSR Sargent et al. 2004

  • Octoploid

    • AFLP Lerceteau-Köhler et al., 2003

Need for a robust, transferable marker for the octoploid, cultivated strawberry!


History of strawberry l.jpg

History of strawberry

1759 “F. ananassa”

1765 Duchesne F. ananassa’s parents

1714


Objetives l.jpg

Objetives

  • Characterize 35 gene-pair haplotype loci

    • Sequence polymorphic alleles

    • Test feasibility to establish relationships between diploid versus octoploid alleles

    • Develop a fingerprint strategy for strawberry cultivars

  • Establish a DNA extraction protocol for strawberry

  • Research phylogenetic relationships between strawberry cultivars by plastid markers


8 ploid strawberries l.jpg

8-ploid strawberries

1600’s

chiloensis

virginiana

iturupensis


Octoploid strawberries l.jpg

Octoploid strawberries

1759 “F. ananassa”

1765 F. ananassa’s parentage proposed

chiloensis

virginiana

1714


2x 4x 6x strawberries l.jpg

gracilis

daltoniana

vesca

nilgerrensis

nubicola

viridis

iinumae

mandshurica

yezoensis

moupinensis 4x

nipponica

orientalis 4x

pentaphylla

moschata

2x,4x, 6x strawberries

2x

6x

4x


Diploids used in this study l.jpg

vesca

nilgerrensis

nubicola

viridis

iinumae

mandshurica

Diploids used in this study

2x


The cultivated strawberry fragaria x ananassa is an octoploid 2n 8x 56 l.jpg

The cultivated strawberry, Fragaria x ananassa, is an octoploid (2n=8x=56)

Genomic complexity: AAA’A’BBB’B’ (Bringhurst, 1990)

Small basic (x=7) genome: C < 200 Mb


Structural genomics in fragaria x ananassa denise cristina manfrim tombolato20 l.jpg

Structural genomics in Fragaria x ananassaDenise Cristina Manfrim Tombolato

  • DNA extraction from a recalcitrant plant species

  • Gene-Pair Haplotypes: novel, complex markers for development of linkage association in a polyploid

  • Development of a fingerprinting strategy for cultivated strawberry

  • Phylogenetic relationships between strawberry cultivars revealed by hypervariable plastid markers


Possible remedy cultivar improvement l.jpg

Possible Remedy = Cultivar improvement

  • Traditional strawberry breeding

    • Linkage mapping in the octoploid background is difficult

  • Molecular approaches

Structural genomics

Molecular markers for MAS


Strawberry genomics at uf l.jpg

Strawberry Genomics at UF

Functional genomics

Structural genomics

Molecular markers for MAS

Gene discovery and analysis of expression

Assessment of

gene function

in transgenic

strawberry


Strawberry genomics at uf23 l.jpg

Strawberry Genomics at UF

Functional genomics

Structural genomics

Molecular markers for MAS

Gene discovery and analysis of expression

Assessment of

gene function

in transgenic

strawberry


Dna extraction from a recalcitrant plant species l.jpg

DNA extraction from a recalcitrant plant species

Plants reported as recalcitrant to DNA extraction

  • Cotton

  • Sugarcane

  • Tomato

  • Grapevine

  • Conifers

  • Chestnut rose

  • Strawberry


Dna extraction protocols l.jpg

DNA extraction protocols

  • 1953: Jones observed differential precipitation of CTAB

    • high ionic strength solutions (>0.7M NaCl), CTAB complexes with proteins and basic and neutral polysaccharides

    • low ionic strength, CTAB precipitates nucleic acids and acidic polysaccharides, leaving proteins and neutral sugars in solution

  • 1980: Murray and Thompson

    • lyophilized plant material

  • 1987: Doyle and Doyle

    • fresh plant material

    • more concentrated buffer to compensate for water in tissue


My 30 attempts to extract dna l.jpg

My 30 attempts to extract DNA

  • Fresh and lyophilized tissue

  • Tissue-to-buffer ratio:

    • from 1.5mg/ml to 500mg/ml


My 30 attempts to extract dna27 l.jpg

My 30 attempts to extract DNA

  • Murray and Thompson, with no CsCl gradient

    • Precipitation by

      • differential ionic strength

      • addition of alcohol at high ionic strength

    • [CTAB]: 1-6%

  • Pre-extraction buffers containing sorbitol and/or PEG

  • Guanidine thiocyanate, with and without CTAB

  • DNAzol kit

  • Urea

  • Differential precipitation by butoxyethanol

  • Isolation of nuclei prior to DNA extraction


Nuclei isolation l.jpg

Nuclei isolation


Dna extraction following nuclei isolation l.jpg

DNA extraction following nuclei isolation

  • SDS

    • 1%

    • 2%

    • 5%, with TIPS and PAS

  • CTAB

    • 2%

  • Qiagen Kit

  • Guanidine thiocyanate


Deproteination of dna l.jpg

Deproteination of DNA

  • Phenol:chloroform

    • Kirby, 1957

  • Sodium perchlorate

    • Wilcockson, 1973


Results l.jpg

Results

  • Mostly low to moderate yields, including post-nuclei isolation methods

  • High DNA yields = non-digestable DNA

    • Guanidine thiocyanate protocols

    • Butoxyethanol


Results32 l.jpg

Results


Results33 l.jpg

Results


Results34 l.jpg

Results

a undigested and supposedly digested samples had smeary aspect, though with most DNA as high molecular weight, in 0.8% agarose gel

b no amplicons observed after PCR with primers for 18S ribosomal DNA

c undigested and allegedly digested samples showed a single high molecular band in 0.8% agarose gel

d determined by spectrophotometer reading of absorption at 260nm; values are a linear extrapolation of milligrams of tissue in fact used for extraction

e tissue-to-buffer ratios were 15mg/ml and 1.5mg/ml

f newly expanded and non-expanded leaflets, respectively

g quantification in agarose gel did not reflect spectrophotometer reading

h loading dye ran to negative pole


Results35 l.jpg

Results


Results36 l.jpg

Results

Precipitation by differential ionic strength versus ethanol


Future attempts manning protocol l.jpg

Future attempts - Manning protocol

  • Extraction buffer

    • Boric acid (BH3O3) to adjust pH of Tris buffer

      • At pH 7.6, boric acid complexes with polyphenols and carbohydrates

  • DNA Precipitation by butoxyethanol

    • 40% volume butoxyEtOH precipitates sugars

    • Equal volume butoxyEtOH precipitates DNA & RNA

    • [Na] = 80mM


Future attempts manning ctab guanidine l.jpg

Future attempts - Manning/CTAB/Guanidine

  • Murray and Thompson and guanidine protocols with precipitation by butoxyethanol?

  • Test different Na concentrations?


Gene pair haplotypes novel complex markers for development of linkage association in a polyploid l.jpg

Gene-Pair Haplotypes: novel, complex markers for development of linkage association in a polyploid


Uses of molecular markers40 l.jpg

Uses of Molecular Markers

  • Genetic characterization of plants and animals

    • Conservation of germplasm

    • Insights on evolution

    • Proprietary issues

  • Marker-assisted selection

  • Linkage maps

    • Map-based positional cloning

    • Comparative mapping

      • Detection of chromosome rearrangements between related taxa


Mapping in a polyploid background l.jpg

Mapping in a polyploid background

Autoploids

Banana 3n=33

Alfalfa 2n=4x=32

Corn 2n=4x=20

Sowerbaea

In an alloploid plant, the number of possible genotypes for one locus with eight different alleles is


Mapping in a polyploid background42 l.jpg

Mapping in a polyploid background

Autoploids

Alloploids

Banana 3n=33

Alfalfa 2n=4x=32

Corn 2n=4x=20

Sowerbaea

Wheat 6x(Kam-Morgan et al. 1989)

Oat 6x(O’Donoughue et al., 1992)

Sugarcane 2n=100 to 130(Arruda, 2001)

In an alloploid plant, the number of possible genotypes for one locus with eight different alleles is


Mapping of strawberry l.jpg

Mapping of strawberry

  • Diploid Fragaria vesca Davis and Yu, 1997

    • SSR markers added to map Sargent et al., 2004

  • Octoploid, AFLP markers Lerceteau-Köhler et al., 2003


Markers maps in strawberry l.jpg

Markers/Maps in strawberry

  • Linkage: fruit color & Sdh isozyme locus Williamson et al. 1995

    • F. vesca ‘Alpine’ varieties

      • Yellow Wonder x Baron Solemacher

  • Linkage: runnering & Pgi-2 isozyme locus Yu & Davis1995


Slide45 l.jpg

  • Rpf1 = resistance gene Haymes et al. 1997

    • RAPD, bulked segregant analysis

  • Gene-specific primers for proteins involved in the anthocyanin biosynthesis pathway and linkage to ‘c’ locus Deng and Davis 2001

    • Northern California F. vesca x F. vesca ‘Alpine’ YW

    • F. vesca ‘Alpine’ YW x F. nubicola

    • PCR-amplified intron length polymorphism of candidate genes

  • SSR octoploid Nourse et al. 2002

  • SSR F. virginiana Ashley et al. 2003

  • SSR F. vesca James et al. 2003

  • SSR F. viridis Sargent et al. 2003

  • SSR from vesca to 8 vescas, ananassa, nubicola, mandschurica, viridis, iinumae, nilgerrensis Monfort 2005

  • 2003 Arnau et al. Inter Simple Sequence Repeat (ISSR) to ID cultivars

  • ISSR-derived SCAR markers Albani et al. 2004

  • SSR in Fragaria Hadonou et al. 2004

  • SSR F. vesca Hadonou et al. 2004

  • SSR Lewers et al. 2005

  • SCAR Haymes et al. 2000

  • SCAR Albani et al. 2004

  • SCAR Davis et al. 1995


Markers in strawberry l.jpg

Markers in strawberry

  • For cultivar identification

    • Isozymes

      • Arulsekar et al. 1981

      • Bringhurst et al. 1981

      • Nehra et al. 1991

      • Bell and Simpson, 1994

    • RAPD

      • Gidoni et al. 1994

      • Hancock et al. 1994

      • Levi et al. 1994

      • Parent & Page, 1995

      • Graham et al. 1996

      • Landry et al., 1997

      • Harrison et al. 1997

      • Degani et al. 1998

      • Degani et al. 2001

    • AFLP

      • Degani et al. 2001


Strawberry linkage maps l.jpg

Strawberry Linkage Maps

  • First strawberry linkage map Davis and Yu 1997

    • RAPD markers, including codominant ones

    • F. vesca ‘Alpine’ Baron Solemacher x WC6 (wild, NH)

  • Gene-specific primers for proteins involved in the anthocyanin biosynthesis pathway Deng and Davis 2001

  • AFLP Lerceteau-Kohler et al. 2003

  • Microsatellite Sargent et al. 2004

    • F. vesca f. semperflores x F. nubicola

    • SSR markers from a gDNA library enriched for SSR

      • Library enrichment Edwards, 1996:

        • Hybridize synthetic SSRs to a membrane

        • Restriction-digest gDNA

        • Attach adaptors to DNA cut ends

        • Hybridize

        • Elute bound DNA

        • PCR-enrich DNA fragments by using primers for the adaptors


Phytophthora fragariae red stele root rot l.jpg

Phytophthora fragariae red stele root rot

  • 5 race-specific plant resistance genes

  • 5 avirulent genes

  • Interact in gene-for-gene system

  • Resistance gene Rpf1 shown to segregate monogenically by use of RAPD markers linked to Rpf1 Haymes 1997


F vesca linkage map l.jpg

F. vesca linkage map


Slide50 l.jpg

AFLP linkage map of the female parent

AFLP linkage map of the male parent


Microsatellite l.jpg

Microsatellite


Slide52 l.jpg

  • F. vesca

    • Cultivars, formae

      • ‘Alpine’ = seasonal

        • Varieties:

          • Yellow Wonder

          • Baron Solemacher

      • f. semperflorens =


Mapping of strawberry53 l.jpg

Mapping of strawberry

  • Diploid Fragaria vesca Davis and Yu, 1997

    • SSR markers added to map Sargent et al., 2004

  • Octoploid, AFLP markers Lerceteau-Köhler et al., 2003

Need for a robust, transferable marker for the octoploid, cultivated strawberry!


Slide54 l.jpg

  • Federova 1946 AABBBBCC

  • Disomic segregation

    • Longley 1926

    • Powers 1944

    • Byrne & Jelenkovic 1976

    • Arulsekar et al. 1981 - 2 isozymes inheritance in octoploid

    • Bringhurst 1990

    • Galleta & Maas 1990

    • Ashley et al. 2003 4 microsatellites segregation disomic in 2 families generated by cross between 4 plants of F. virginiana

  • Autoalloploid

    • Senanayake & Bringhurst 1967 AA A’A’ BB BB; vesca and viridis as donators of “A” genome

    • Lerceteau-Kohler 2003


Evidences supporting vesca as ancestor l.jpg

Evidences supporting vesca as ancestor

  • Millardet, 1894

    • White-fruited F. vesca x red-fruited F. chiloensis

    • “False hybrids”

  • Ichijima, 1926

    • F. bracteata x F. virginiana

    • 7 bivalents, 21 univalents

  • Mangelsdorf & East, 1926, criticizing Milladert

    • F. vesca x F. chiloensis

    • F. vesca x F. virginiana

  • Bringhurst and Gill, 1970

    • Observed natural hybrids between F. chiloensis and F. vesca

  • 94% SSRs developed for vesca amplified ananassa fragments Monfort 2005


C values determined by flow cytometry l.jpg

C-values determined by flow cytometry

  • Nehra et al. 1991

    • F. x ananassa 0.61pg

  • Antonius and Ahokas 1996, cited by Bennett et al 2000

    • F. viridis 0.10pg ~ 105Mbp

    • F. moschata 0.35pg ~ 686Mbp

    • F. virginiana 0.40pg ~ 784Mpb

  • Akiyama et al. 2001, using Arabidopsis rather than chicken erythrocytes as comparison

    • F. vesca 164Mbp


Gene pair haplotype l.jpg

Gene-Pair Haplotype

Complex Molecular Marker to

  • study segregation of the diploid subgenomes in the octoploid background, thus permitting molecular mapping


Gene pair haplotype58 l.jpg

Gene-Pair Haplotype

Complex Molecular Marker to

  • study segregation of the diploid subgenomes in the octoploid background, thus permitting molecular mapping


Gene pair haplotype59 l.jpg

InDel

RFLP

Gene-Pair Haplotype

Complex Molecular Marker to

  • study segregation of the diploid subgenomes in the octoploid background, thus permitting molecular mapping


Gene pair haplotype60 l.jpg

Gene-Pair Haplotype

Complex Molecular Marker to

  • study segregation of the diploid subgenomes in the octoploid background, thus permitting molecular mapping

  • delineate allele contribution by diploid genomes to the octoploid genome


Gene pair haplotype61 l.jpg

SNP, SSR

Gene-Pair Haplotype

Complex Molecular Marker to

  • study segregation of the diploid subgenomes in the octoploid background, thus permitting molecular mapping

  • delineate allele contribution by diploid genomes to the octoploid genome


Gene pair haplotype62 l.jpg

InDel

RFLP

SNP, SSR

Gene-Pair Haplotype

Complex Molecular Marker to identify polymorphisms

  • between subgenomes

  • within subgenomes


Gene pair haplotype63 l.jpg

Gene 1

Gene 2

Gene-Pair Haplotype

octoploid

strawberry

C

TATATA

BamHI

T

TATA

BamHI

T

EcoRI

TATATA

C

TATA

BamHI

InDel

SNP

SSR

RFLP


Gene pair haplotype64 l.jpg

Gene 1

Gene 2

Gene-Pair Haplotype

octoploid

strawberry

C

TATATA

BamHI

T

Within genomes

TATA

BamHI

T

EcoRI

TATATA

Between

genomes

C

TATA

BamHI

InDel

SNP

SSR

RFLP


How do i find the genes that compose a pair l.jpg

How do I find the genes that compose a pair?

  • Exploring potential microcolinearity between strawberry and arabidopsis

  • Using fosmid sequence to predict genes’ location within the sequence

    • Gene prediction softwares

    • Comparing fosmid sequences to existing sequences in the bank (Blast)


Microcolinearity l.jpg

At5g52950

At5g52960

EST”a”

EST”x”

Microcolinearity

311bp

arabidopsis

octoploid

strawberry

Primer F

Primer R


Microcolinearity67 l.jpg

Microcolinearity

  • Using the Genome Database for Rosaceae (GDR)

    • 1505 strawberry ESTs vs arabidopsis

      • 9 were potentially useful due to putative adjacency

        • only 2 have worked, generating PCR products that corresponded to the target sequence: GPH5, GPH23

  • Using FASTA

    • 250 strawberry sequences

      • 36 potential pairs

        • GPH30, 56

    • useless pairs

      • > 3.5kb apart in arabidopsis

      • a single strawberry EST similar to 2 neighboring arabidopsis genes (gene families)

      • sequences similar in FASTA but not in Blast

      • pairs for which primers were designed, but there was no amplicon

        • no microcolinearity

        • microcolinearity exists, but gene orientations are not kept


Slide68 l.jpg

GPHs

2, 3, 4, 5*, 6, 10*, 20, 21, 22, 23*, 27, 30, 31, 56

  • GPHs cloned, sequenced

  • no amplicon generated

  • under PCR optimization

  • new GPHs cloned, sequenced

underlined: FASTA approach

italicized: fosmid

* : sequence corresponded to gene pair


Gph23 l.jpg

GPH23

  • Expect 982bp, based on arabidopsis intergenic space

  • Pair:

    • Oxidoreductase

    • DNA-binding protein-related

  • Amplified

    • Strawberry Festival

    • F. iinumae

    • F. mandschurica

  • Probe for F. vesca

    • I designed primers internal to gene for DNA-binding protein-related

    • Amplified, cloned, sent to sequence

      • awful qualify!


Gph2370 l.jpg

GPH23


Gph2371 l.jpg

GPH23


Gph2372 l.jpg

GPH23


Slide73 l.jpg

GPH5

  • Expect 2196bp, based on arabidopsis intergenic space

  • Pair:

    • Glycosyl hydrolase

    • Glycosyl transferase


Slide74 l.jpg

GPH5


Gph5 no similarity w est in b end l.jpg

GPH5 - no similarity w/ EST in B end


Gph30 l.jpg

GPH30

  • Expect 1.8kb

  • Pair:

    • ferredoxin-related

    • human Rev interacting protein

  • Tested

    • 51.4C, 54, 56.4, 60, 60.3

    • Phil’s and Dawn’s Festival preps

  • Results

    • Non-specific bands at 51.4C and 56.4

    • 2 bands at 54C ~1.8kb?, 1kb?, same intensity

    • No product at >60C


Gph56 l.jpg

GPH56

  • Expect 3kb

  • Pair:

    • At: fructose bisphosphate; Fa: aldolase

    • At: glycosyl hydrolase; Fa: 1,4-glucanase

  • Tested: 51.4, 55, 56.4, 59, 60.3, 63

  • Result:

    • 55C: 4 bands (5, 4, 2, 1.5kb)

    • 56.4: 3 bands (3.5, 3, 2kb)

    • 51.4C, 59, 63: no bands

    • 60.3C: 3.5kb


A second approach to detect gene pairs l.jpg

A second approach to detect gene pairs

  • Received 9 Fosmid sequences

    • 30-40kb each


Gene prediction softwares l.jpg

AAT:   Analysis and Annotation Tool

DioGenes:  Find protein-encoding regions in genomic sequences

Doublescan: Comparative prediction of protein coding genes

FGENESH:  Splice sites, protein coding exons & gene models

FirstEF: First-exon & promoter prediction program 

GATG ORF finder: Gene Prediction Using GA/TG dinucleotides

GeneID:   Gene identification and structure prediction 

GeneMark:  Markov model with training datasets to predict genes

GeneParser2:    Identification of protein coding regions 

Generation:   Microbial gene prediction system

GeneSeqer: Identify exon/intron structure in pre-mRNA

Genie:   Gene finder based on hidden Markov models

GenLang:   Linguistics based method to find genes

GenomeScan: Based on GenScan incorporating protein homology

GenScan:   Identification of gene structures in genomic DNA

GenViewer:   Predicting of protien-coding gene structures

GlimmerM:   A system for finding genes in microbial DNA 

Grail:    DNA sequence analysis tool

HMMGene: Prediction of vertebrate and C. elegans genes

MetaGene: submit sequences to seven gene prediction engines 

MORGAN:    A decision tree system for finding genes

MZEF:   Predict protein coding exons in genomic DNA

NetGene2:   Neural network predictions of splice sites

ORF Finder:   Search for open reading frame, at NCBI 

Pombe:  Predict protein coding exons fission yeast

Procrustus:   Gene recognition via spliced alignment

SLAM:  Comparative gene annotation and alignment

SplicePredictor: Identify potential splice sites in plant pre-mRNA

SpliceSite Prediction:   Splice site prediction by neural network

Twinscan:  Comparative gene prediction

VEIL:   A hidden Markov model for finding genes

Wise2 :  Intelligent algorithm for DNA searches

WebGene: Several tools for prediction and analysis gene structures

Xgrail:   Find exons and other features

YeastGene: Measure the coding ability of an ORF in S cerevisiae

Gene prediction softwares

Software must be sensitive and specific


Gene prediction softwares80 l.jpg

AAT:   Analysis and Annotation Tool

DioGenes:  Find protein-encoding regions in genomic sequences

Doublescan: Comparative prediction of protein coding genes

FGENESH:  Splice sites, protein coding exons & gene models

FirstEF: First-exon & promoter prediction program 

GATG ORF finder: Gene Prediction Using GA/TG dinucleotides

GeneID:   Gene identification and structure prediction 

GeneMark:  Markov model with training datasets to predict genes

GeneParser2:    Identification of protein coding regions 

Generation:   Microbial gene prediction system

GeneSeqer: Identify exon/intron structure in pre-mRNA

Genie:   Gene finder based on hidden Markov models

GenLang:   Linguistics based method to find genes

GenomeScan: Based on GenScan incorporating protein homology

GenScan:   Identification of gene structures in genomic DNA

GenViewer:   Predicting of protein-coding gene structures

GlimmerM:   A system for finding genes in microbial DNA 

Grail:    DNA sequence analysis tool

HMMGene: Prediction of vertebrate and C. elegans genes

MetaGene: submit sequences to seven gene prediction engines 

MORGAN:    A decision tree system for finding genes

MZEF:   Predict protein coding exons in genomic DNA

NetGene2:   Neural network predictions of splice sites

ORF Finder:   Search for open reading frame, at NCBI 

Pombe:  Predict protein coding exons fission yeast

Procrustus:   Gene recognition via spliced alignment

SLAM:  Comparative gene annotation and alignment

SplicePredictor: Identify potential splice sites in plant pre-mRNA

SpliceSite Prediction:   Splice site prediction by neural network

Twinscan:  Comparative gene prediction

VEIL:   A hidden Markov model for finding genes

Wise2 :  Intelligent algorithm for DNA searches

WebGene: Several tools for prediction and analysis gene structures

Xgrail:   Find exons and other features

YeastGene: Measure the coding ability of an ORF in S cerevisiae

Gene prediction softwares


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Comparison ofgene prediction softwares


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Development of a fingerprinting strategy for cultivated strawberry


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Molecular Markers for Identification of Plant Cultivars

  • Plant Variety Protection

    • Description of morphological characteristics

    • PCR-based markers: fast method

    • “The genetic anatomy of a patented yellow bean” - Pallottini et al. 2004

  • Molecular markers used in strawberry

    • AFLP Degani et al. 2001

    • RAPD Zebrowska and Tyrka 2003; Zhang et al. 2003

    • RAHFP Martelli et al. 2002

    • CAPS Kunihisa et al. 2005


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Cultivar ID

  • Isozymes

    • Electrophoretic characterization of strawberry cultivars

      • RS Bringhurst, S Arulsekar, JF HANCOCK, V VOTH - Journal of the American Society for Horticultural Science, 1981 . 106: 684-687


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GPH23

  • Amplified a ~2.2kb band of

    • Strawberry Festival

    • Carmine

    • Diamante

    • Rosa Linda

    • Sweet Charlie

    • Treasure

    • USDA4809

    • LF9

    • F. iinumae

    • F. mandschurica

  • Digested 200ul of amplicon with HaeIII

  • Precipitated with ethanol

  • Resuspended in 10ul water

  • Ran digested product in a Agilent Bioanalyzer


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GPH23/HaeIII


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GPH23/HaeIII

Strawberry Festival

Sweet Charlie

F. mandschurica

Rosa Linda

Undigested

Diamante

USDA4809

Treasure

F. iinumae

Carmine

Ladder

EcoRI

LF9


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GPH23/HaeIII

  • Inconclusive results

    • Similar pattern between Rosa Linda and Sweet Charlie expected

    • Not expected: LF9 completely different from its progenitor, Strawberry Festival!

  • Repeat this technique with a different gene pair


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1759 - Philip Miller first cited ananassa in the Gardner’s Dictionary,

1765- Duchesne states his suspicions on F. ananassa’s parents

1714 - because of the fruit’s big size, Fresier takes “frutillar” to France, when returning from expedition sent by Louis XIV


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A model of a plant gene

Promoter: 2-12 cis-elements

ATG

CAAT

TATAAA

75bp from ATG

10-30bp

10-1000bp

Locus control region

(Cis-acting sequence that organizes a gene cluster into an active chromatin block and enhances transcription.)


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