Marker devt. workshop 27022012
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Transcript of Marker devt. workshop 27022012
Marker development for mappingRavi Koppolu
RG: Plant ArchitectureIPK, Gatersleben
Outline
Few concepts
Different types of molecular markers and applications
Marker development for gene mapping
Exon 1
5‘ 3‘
5‘-UTR
Start codon:ATG
Intron 1Promoter3‘-UTR
I 2
Stop codon:TAGTAATGA
Exon 3
Genomic DNA (gDNA) mRNA Protein
Exon 2genomic DNA
Transcr. Start
Start:ATG
Stop:TAGTAATGA
ORF (open reading frame) coding sequence
Translation
Protein
Poly(A)A..Amature mRNA
Transcription & Post transcriptional modification
Slide from Dr. Thorsten Schnurbusch
Polymorphism?Is the ability to distinguish two or more individuals
The variation shown here is due to four different alleles at a particular gene.
Molecular markers can identify sequence polymorphisms among two or more individuals which may result in the change of phenotype.
vrs4.k TGGTTGCAGCGGCCACGACACCGGGGGC-GGGGCGCCGTGCGCTGCGTG :Mutant allele MFB104 TGGTTGCAGCGGCCACGACACCGGGGGCCGGGGCGCCGTGCGCTGCGTG :Wild allele
2-rowed spike 6-rowed spike
Subsequently, genetic variation at DNA aroused
due to mutation will cause variation in protein
Protein markers
MutationMutation arises genetic
variation at the DNA levelDNA
markers
A sequence of DNA or protein that can be screened to reveal key attributes of its state or composition and thus used to reveal genetic
variation
Molecular marker
Genomic DNA mutations can be classified into
GATCCGAGTATCGCAATTAGCAGATCCGAGTGTCGCAATTAGCA
Base substitution
GATCCGAGTATCGCATGCATTAGCAGATCCGAGTA 7 7 7 7 7 7 7 7 7 ATTAGCA
Deletion
GATCCGAGTATCGCAATTAGCAGATCCGAGTATCGCAGCATTAGCA
Insertion
GATCCGAGTATCGCAATTAGCAGATCCGAGTATCTCGCAATTAGCA
Duplication
GATCCGAGTATCGCAATTAGCAGATGCCAGTATCGCAATTAGCA
Inversion
Molecular marker typesCo-dominant marker systemsRestriction Fragment Length Polymorphisms (RFLPs)
Simple sequence repeats (SSRs)
SNP based marker systems
Dominant marker systemsRandom amplified polymorphic DNAs (RAPD)
Amplified fragment length polymorphism (AFLP)
Diversity arrays technology (DArT)
Co-dominant vs. Dominant marker systems
Collard et al. 2005; Euphytica
Co-dominant marker Dominant marker
Co-dominant markers follow Mendelian inheritance pattern
Simple sequence repeats (SSRs)SSRs contain tandem repeated single, double, triple nucleotides several times
Allelic variants differ in terms of number of repeats
Single nucleotide polymorphisms (SNPs) A mutation that causes single base change is SNP Most of them don't have a phenotypic effect SNPs can be alleles of a particular gene SNPs are most abundant types of DNA variation found among individuals
of same species
Applications of molecular markers
Germplasm identification, Classification Genetic diversity/Relatedness Selection of parents for making wide crosses Development of molecular linkage maps Marker assisted selection Genomic selection Map-Based cloning of genes
Synteny enables to recruit markers from related species
Close et al. 2010; BMC GenomicsMayer et al. 2011, Pl. Cell
BLAST against barley EST’s
Flank introns/UTRs with primers
viroBLAST against barley
contigs
Resequence
SNP
Length polymorphism
CAPS
dCAPS
Genotyping on Agarose
Syntenic/Colinear regions
Marker development procedure at AG PBP
Resources for our Marker development workshop
Genome ZippersProvide information on putative linear gene order for 86% barley genes based on synteny ̴with rice, sorghum and Brachypodium.
Mayer et al. Plant Physiol, 2009 Available athttp://mips.helmholtz-muenchen.de/plant/triticeae/barleyDisclaimerGZ.jsp
A snapshot of Genome Zipper
Syntenic cereal sequence databases
http://rice.plantbiology.msu.edu/annotation_pseudo_current.shtml
http://www.brachypodium.org/
Barley viroBLAST
Barley viroBLAST contains sequence data like
1.) 28X Illumina shot gun reads based on Morex2.) Sequenced BACs3.) Sorted chromosome sequences4.) Full length cDNA sequences
http://webblast.ipk-gatersleben.de/barley/index.php
BLAST output with the barley viroBLAST sequence as query against barley EST database
The sequence/s from viroBLAST can be BLASTed against barley ESTs to identify Exon-Intron boundaries
Primer design Primers designed to span boundaries of two exonic regions or to the UTRs Primers can be picked manually or by
using software’s like primer3
GC content of a primer pair should better be >=30%
Avoid having multiple A’s or T’s at the 3’end.
Check whether primers are forming any secondary structures or dimers
Better to have overlapping Tm for different primer pairs
If designed manually don't forget to reverse complement the reverse primer
Stem loops Dimers
Resequencing of primer pairs
Resequence the primer pairs in the parents of interest to identify sequence polymorphisms.
Sequence analysis will be demonstrated during the workshop
Snapshot showing SNPs and deletions identified after resequencing in parents
Development of CAPS and dCAPS markersCAPS: Cleaved Amplified Polymorphic sequence
In principle CAPS is similar to RFLP except that a shorter PCR amplified product with known SNPs is digested with restriction enzymes instead of whole genomic DNA.
Contd….
dCAPS: Derived Cleaved Amplified Polymorphic sequence
In dCAPS assay mismatches in PCR primers are introduced to create restriction endonuclease polymorphism based on the target mutation.
BclI recognition site: CC(N)7GG
Web links for Marker developmentBrachypodium database: http://www.brachypodium.org/
Rice databasehttp://rice.plantbiology.msu.edu/annotation_pseudo_current.shtml
Rice ID Converterhttp://rapdb.dna.affrc.go.jp/tools/converter
Barley viroBLASThttp://webblast.ipk-gatersleben.de/barley/viroblast.php
NCBI BLASThttp://blast.ncbi.nlm.nih.gov/Blast.cgi?CMD=Web&PAGE_TYPE=BlastHome
IDT manual Primer designhttp://eu.idtdna.com/analyzer/Applications/OligoAnalyzer/Default.aspxhttp://www.basic.northwestern.edu/biotools/oligocalc.html
Primer3http://frodo.wi.mit.edu/primer3/
Reverse complement generatorhttp://www.bioinformatics.org/sms/rev_comp.html
Clustalwhttp://www.ebi.ac.uk/Tools/msa/clustalw2/
Nebcutterhttp://tools.neb.com/NEBcutter2/
dCAPS finderhttp://helix.wustl.edu/dcaps/dcaps.html