Recitation 5 Molecular Markers

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Recitation 5Molecular Markers

• CSS/HORT 430
• Plant Genetics

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What are molecular markers?

• Genetic information resides in the genome
• A molecular marker is a known DNA sequence.
• It can be described as a variation, which may
  arise due to mutation or alteration in the
  genomic loci, that can be observed.

gtataagtgaaccactcagggtcctggccgggcacagtggctcacgcct
gtaatcccagcctttgggaggccgaggtgggtggatcatgaggtcaggag
ttcaagaccagcctggccaaatggcgaaacattgtctctactaaaagtac
aaaaattagccagacgtggtggtgctcactgtaatcccagctactcggga
ggctgaggcaggaaaatcacttgaacccgggaggcggaggtcacagttag
ccaagatcacaccactgtactccag
ATP5L2    ATP synthase C22orf11   chromosome 22
ORF PANX2  pannexin 2
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Characteristics of Ideal Polymorphic Markers

• Co-dominant (distinguish homozygotes and
  heterozygotes)
• Nondestructive assay
• Complete penetrance
• Early onset of phenotypic expression
• High polymorphism
• Random distribution throughout the genome
• Assay can be automated

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Phenotypic markers
2-rowed 6-rowed
Black white
non-waxy waxy
hulled naked
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Conversion of phenotypic marker to molecular
marker
non-waxy
How?
waxy
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Methods for marker development

• Restriction digest
• Polymerase chain reaction (PCR)
• Sequence analysis

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RFLP - Restriction fragment length polymorphism
http//www.usask.ca/agriculture/plantsci/classes/p
lsc416/projects_2002/pawlin/resources/rflp4.jpg
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Polymerase Chain Reaction
http//fig.cox.miami.edu/cmallery/150/gene/c7.20.
7.pcr.jpg
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Marker development through Sequence analysis

• Find the candidate gene (literature)
• Get the sequence of the candidate gene (GenBank)
• Determine sequence polymorphism between the
  parents (alignment)
• Develop PCR marker (primer design)
• Screen the mapping population (PCR,
  electrophoresis)
• Score segregation (gel photo)

Lets get some practical experience
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Things to do in this class

• Search sequences from Database
• Non-waxy (dominant Waxy) allele AB088761
• Waxy (recessive waxy) allele AB087716
• Alignment of sequences for polymorphism
• ClustalW
• Design primers for PCR based screening
• Primer3

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Population screening

• Primer design
• Forward primer CAAACAGACGACAAGCGGAGAA
• Reverse primer TAGAAAAAGAAAACATCAAGCA
• Parent screening
• Dominant parent (WaxyWaxy) 1000 bp
• Recessive parent (waxywaxy) 600 bp

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Question? How will you find markers linked to EFB
resistance in Hazelnut
Repulsion
Coupling
RP- Resistant Parent SP- Susceptible
Parent R1,R2,R3- Resistant Seedlings S1,S2,S3-
Susceptible Seedlings