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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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challenges during strawberry production
Challenges during strawberry production
  • Diseases - Florida: anthracnose
  • Pests
challenges for florida strawberry production
Challenges for Florida strawberry production
  • Diseases
  • Pests
  • Phase-out of methylbromide
  • Market competition/consolidation
one remedy plant improvement
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
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
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
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
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
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
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
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
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
History of strawberry

1759 “F. ananassa”

1765 Duchesne F. ananassa’s parents

1714

objetives
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
8-ploid strawberries

1600’s

chiloensis

virginiana

iturupensis

octoploid strawberries
Octoploid strawberries

1759 “F. ananassa”

1765 F. ananassa’s parentage proposed

chiloensis

virginiana

1714

2x 4x 6x strawberries

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

vesca

nilgerrensis

nubicola

viridis

iinumae

mandshurica

Diploids used in this study

2x

the cultivated strawberry fragaria x ananassa is an octoploid 2n 8x 56
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
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
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
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
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
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
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
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
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
dna extraction following nuclei isolation
DNA extraction following nuclei isolation
  • SDS
    • 1%
    • 2%
    • 5%, with TIPS and PAS
  • CTAB
    • 2%
  • Qiagen Kit
  • Guanidine thiocyanate
deproteination of dna
Deproteination of DNA
  • Phenol:chloroform
    • Kirby, 1957
  • Sodium perchlorate
    • Wilcockson, 1973
results
Results
  • Mostly low to moderate yields, including post-nuclei isolation methods
  • High DNA yields = non-digestable DNA
    • Guanidine thiocyanate protocols
    • Butoxyethanol
results34
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

results36
Results

Precipitation by differential ionic strength versus ethanol

future attempts manning protocol
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
Future attempts - Manning/CTAB/Guanidine
  • Murray and Thompson and guanidine protocols with precipitation by butoxyethanol?
  • Test different Na concentrations?
uses of molecular markers40
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
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
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
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
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
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
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
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
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
slide50

AFLP linkage map of the female parent

AFLP linkage map of the male parent

slide52
F. vesca
    • Cultivars, formae
      • ‘Alpine’ = seasonal
        • Varieties:
          • Yellow Wonder
          • Baron Solemacher
      • f. semperflorens =
mapping of strawberry53
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
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
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
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
Gene-Pair Haplotype

Complex Molecular Marker to

  • study segregation of the diploid subgenomes in the octoploid background, thus permitting molecular mapping
gene pair haplotype58
Gene-Pair Haplotype

Complex Molecular Marker to

  • study segregation of the diploid subgenomes in the octoploid background, thus permitting molecular mapping
gene pair haplotype59

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
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

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

InDel

RFLP

SNP, SSR

Gene-Pair Haplotype

Complex Molecular Marker to identify polymorphisms

  • between subgenomes
  • within subgenomes
gene pair haplotype63

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

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
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

At5g52950

At5g52960

EST”a”

EST”x”

Microcolinearity

311bp

arabidopsis

octoploid

strawberry

Primer F

Primer R

microcolinearity67
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
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
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!
slide73
GPH5
  • Expect 2196bp, based on arabidopsis intergenic space
  • Pair:
    • Glycosyl hydrolase
    • Glycosyl transferase
gph30
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
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
A second approach to detect gene pairs
  • Received 9 Fosmid sequences
    • 30-40kb each
gene prediction softwares

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

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
molecular markers for identification of plant cultivars
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
cultivar id
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
gph2385
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
gph23 haeiii87
GPH23/HaeIII

Strawberry Festival

Sweet Charlie

F. mandschurica

Rosa Linda

Undigested

Diamante

USDA4809

Treasure

F. iinumae

Carmine

Ladder

EcoRI

LF9

gph23 haeiii88
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
slide89

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

a model of a plant gene
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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