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