Marker development for gene mapping in barley with necessary resources as web links at the end of the presentation.

1. Marker development for
mapping
Ravi Koppolu
RG: Plant Architecture
IPK, Gatersleben

2. Outline
 Few concepts
 Different types of molecular markers and applications
 Marker development for gene mapping

3. Exon 1
5‘ 3‘
5‘-UTR
Start codon:
ATG
Intron 1Promoter
3‘-UTR
I 2
Stop codon:
TAG
TAA
TGA
Exon 3
Genomic DNA (gDNA)  mRNA  Protein
Exon 2
genomic DNA
Transcr.
Start
Start:
ATG
Stop:
TAG
TAA
TGA
ORF (open reading frame) coding sequence
Translation
Protein
Poly(A)A..A
mature mRNA
Transcription & Post
transcriptional modification
Slide from Dr. Thorsten Schnurbusch

4. 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

5. Subsequently, genetic
variation at DNA aroused
due to mutation will cause
variation in protein
Protein
markers
Mutation
Mutation arises genetic
variation at the DNA level
DNA
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

6. Genomic DNA mutations can be classified into
GATCCGAGTATCGCAATTAGCA
GATCCGAGTGTCGCAATTAGCA
Base substitution
GATCCGAGTATCGCATGCATTAGCA
GATCCGAGTA ̶ ̶ ̶ ̶ ̶ ̶ ̶ ̶ ̶ ATTAGCA
Deletion
GATCCGAGTATCGCAATTAGCA
GATCCGAGTATCGCAGCATTAGCA
Insertion
GATCCGAGTATCGCAATTAGCA
GATCCGAGTATCTCGCAATTAGCA
Duplication
GATCCGAGTATCGCAATTAGCA
GATGCCAGTATCGCAATTAGCA
Inversion

7. Molecular marker types
Co-dominant marker systems
Restriction Fragment Length Polymorphisms (RFLPs)
Simple sequence repeats (SSRs)
SNP based marker systems
Dominant marker systems
Random amplified polymorphic DNAs (RAPD)
Amplified fragment length polymorphism (AFLP)
Diversity arrays technology (DArT)

8. Co-dominant vs. Dominant marker systems
Collard et al. 2005; Euphytica
Co-dominant marker Dominant marker
Co-dominant markers follow Mendelian inheritance pattern

10. Simple sequence repeats (SSRs)
SSRs contain tandem repeated single, double, triple nucleotides several times
Allelic variants differ in terms of number of repeats

11. 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

12. 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

13. Synteny enables to recruit markers from related species
Close et al. 2010; BMC Genomics
Mayer et al. 2011, Pl. Cell

14. 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

15. Resources for our Marker development
workshop

16. Genome Zippers
Provide 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 at
http://mips.helmholtz-muenchen.de/plant/triticeae/barleyDisclaimerGZ.jsp
A snapshot of Genome Zipper

17. Syntenic cereal sequence databases
http://rice.plantbiology.msu.edu/annotation_pseudo_current.shtml
http://www.brachypodium.org/

18. Barley viroBLAST
Barley viroBLAST contains sequence data like
1.) 28X Illumina shot gun reads based on Morex
2.) Sequenced BACs
3.) Sorted chromosome sequences
4.) Full length cDNA sequences
http://webblast.ipk-gatersleben.de/barley/index.php

19. 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

20. 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

21. 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

22. Development of CAPS and dCAPS markers
CAPS: 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….

23. 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

24. Web links for Marker development
Brachypodium database:
http://www.brachypodium.org/
Rice database
http://rice.plantbiology.msu.edu/annotation_pseudo_current.shtml
Rice ID Converter
http://rapdb.dna.affrc.go.jp/tools/converter
Barley viroBLAST
http://webblast.ipk-gatersleben.de/barley/viroblast.php
NCBI BLAST
http://blast.ncbi.nlm.nih.gov/Blast.cgi?CMD=Web&PAGE_TYPE=BlastHome
IDT manual Primer design
http://eu.idtdna.com/analyzer/Applications/OligoAnalyzer/Default.aspx
http://www.basic.northwestern.edu/biotools/oligocalc.html
Primer3
http://frodo.wi.mit.edu/primer3/
Reverse complement generator
http://www.bioinformatics.org/sms/rev_comp.html
Clustalw
http://www.ebi.ac.uk/Tools/msa/clustalw2/
Nebcutter
http://tools.neb.com/NEBcutter2/
dCAPS finder
http://helix.wustl.edu/dcaps/dcaps.html