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    Lisa Harper, Curator: USDA ARS PGEC, Albany and University of California, Berkeley, CA

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    Raj Kumar
    This document provides an overview of how to use genetic mapping techniques to identify the sequence of a gene of interest, known as "walking to a gene". It involves making a mapping population, initially mapping the mutant gene to a chromosomal bin, then using additional markers to do fine structure mapping within that bin to progressively narrow down the region containing the gene. Once the region is small enough, sequence differences between wildtype and mutant can be identified to find the gene. Key steps outlined are making a mapping population, initial mapping, finding additional markers from MaizeGDB, fine structure mapping, and then sequencing candidate genes.

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    Lisa Harper, Curator: USDA ARS PGEC, Albany and University of California, Berkeley, CA

    Uploaded by

    Raj Kumar
    67 views137 pages
    This document provides an overview of how to use genetic mapping techniques to identify the sequence of a gene of interest, known as "walking to a gene". It involves making a mapping population, initially mapping the mutant gene to a chromosomal bin, then using additional markers to do fine structure mapping within that bin to progressively narrow down the region containing the gene. Once the region is small enough, sequence differences between wildtype and mutant can be identified to find the gene. Key steps outlined are making a mapping population, initial mapping, finding additional markers from MaizeGDB, fine structure mapping, and then sequencing candidate genes.

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    MaizeGDB_ISU

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    This document provides an overview of how to use genetic mapping techniques to identify the sequence of a gene of interest, known as "walking to a gene". It involves making a mapping population, initially mapping the mutant gene to a chromosomal bin, then using additional markers to do fine structure mapping within that bin to progressively narrow down the region containing the gene. Once the region is small enough, sequence differences between wildtype and mutant can be identified to find the gene. Key steps outlined are making a mapping population, initial mapping, finding additional markers from MaizeGDB, fine structure mapping, and then sequencing candidate genes.

    Copyright:

    Attribution Non-Commercial (BY-NC)
    Download as PPT, PDF, TXT or read online from Scribd
    Download as ppt, pdf, or txt
    67 views137 pages

    Lisa Harper, Curator: USDA ARS PGEC, Albany and University of California, Berkeley, CA

    Uploaded by

    Raj Kumar
    This document provides an overview of how to use genetic mapping techniques to identify the sequence of a gene of interest, known as "walking to a gene". It involves making a mapping population, initially mapping the mutant gene to a chromosomal bin, then using additional markers to do fine structure mapping within that bin to progressively narrow down the region containing the gene. Once the region is small enough, sequence differences between wildtype and mutant can be identified to find the gene. Key steps outlined are making a mapping population, initial mapping, finding additional markers from MaizeGDB, fine structure mapping, and then sequencing candidate genes.

    Copyright:

    Attribution Non-Commercial (BY-NC)
    Download as PPT, PDF, TXT or read online from Scribd
    Download as ppt, pdf, or txt
    of 137

    maizegbd.

    org
    Lisa Harper, curator
    USDA ARS PGEC, Albany and University of California, Berkeley, CA

    Carolyn Lawrence, PI
    Darwin Campbell, database administrator
    Taner Sen, computational biologist
    Carson Andorf, computer scientist
    USDA ARS and Iowa State University, Ames, Iowa

    Mary (Polacco) Schaeffer, curator


    USDA ARS and University of Missouri, Columbia, MO
    Outline of Talk
    1. Brief Introduction- What’s available?
    2. General Features from the MaizeGDB homepage
    3. Searching the Database
    4. Community Curation
    5. Usage Example: Walking to genes
    6. Case Study: Find SSRs close to mac1
    7. Finding QTLs
    8. Finding cytological position from genetic position
    Our Mission
    •Integrate new maize genetic and genomic data into the
    database.
    -Expand mutant and phenotype data and tools.
    -Expand structural and genetic maps.
    -Soon!! Provide a Genome Browser and access to gene models.
    -Compile and make accessible at MaizeGDB the annual Maize
    Newsletter.

    •Provide community support services (e.g., coordinating


    Maize Meeting, MGEC Elections, Polls, etc.).
    Coming Soon
    •POPCORN:
    •A Portal through which you can blast all sources of Maize
    sequences at once.
    •A Mechanism for Data from ANY project to migrate to
    MaizeGDB.
    •Community Gene Annotation (complete with “motivation”).
    •Beefed up Phenotype Tools, Plant Ontology and PO
    annotation.
    •Some automated literature mining.
    Coming Soon: MaizeGDB
    Genome Browser

    *BAC info with links to appropriate MaizeGDB pages


    *FPC contigs clearly showing gaps
    *Marker data with links to MaizeGD
    *Sequence track that shows bases
    *Various Gene models will be available as separate tracks
    Outline of Talk
    1. Brief Introduction- What’s available?
    2. General Features from the MaizeGDB homepage
    3. Searching the Database
    4. Community Curation
    5. Usage Example: Walking to genes
    6. Case Study: Find SSRs close to mac1
    7. Finding QTLs
    8. Finding cytological position from genetic position
    Remember to SCROLL DOWN
    Remember to SCROLL DOWN
    Keep scrolling….
    Scroll Down
    For more
    Goodies!!
    Outline of Talk
    1. Brief Introduction- What’s available?
    2. General Features from the MaizeGDB homepage
    3. Searching the Database
    4. Community Curation
    5. Usage Example: Walking to genes
    6. Case Study: Find SSRs close to mac1
    7. Finding QTLs
    8. Finding cytological position from genetic position
    Remember to
    SCROLL DOWN
    “Advanced” Searches
    Where is lg1 on the genetic map?
    x lg1
    x lg1
    Here is a list of all the genetic maps that contain lg1
    Why the Huge difference??
    Making the Intermated B73 X MO17 mapping population
    B73 Mo17

    Single F1
    plant was
    selfed
    X
    Grow up 200 plants,
    random mating

    Genotype of 5 of those 200 plants


    Intermate these F2s, and you “accumulate recombinations”
    Select 100 ears, pick 5 kernals from each ear. Put in a
    bag, shake, plant, more random matings (2nd generation).
    Repeat, repeat. Intermating was done 4 times.

    From these lines, generate Recombinant Inbred lines by repeated selfing (5x or more)

    x x x x x x
    x x x x x x
    x x x x x x
    x x x x x x
    x x x x x x
    These plants where used for the
    “Intermated B73 Mo17 genetic map
    There are 288 lines in the IBM population. To make the map, MMP project
    researchers mapped thousands of markers. They use primers that amplify a
    different length fragment in Mo17 vs B73.

    B73 Mo17

    EcoR1
    PCR + EcoR1

    Line10
    Mo17
    Line1
    Line2
    Line3
    Line4
    Line5
    Line6
    Line7
    Line8
    Line9
    B73

    So, you gene’s ‘code’ is: MMBBMMBBMB…


    Then, the MMPers figure out, with their computers, where
    that matches in the genome.

    So, you gene’s ‘code’ is: MMBBMM …

    Line 1 Line 2 Line 3 Line 4 Line 5 Line 6

    MMMMMB Nope!

    MBMBMM Nope!

    MMBBMM YEAH!
    Search Phenotypes
    Genes which when mutated have
    liguleless phentoype

    Liguleless alleles

    Stocks
    Sometimes it goes wrong…
    Try spelling out the name, or try a different search criteria,
    Or go to the complete search page
    Any Questions about Searching for Data
    In MaizeGDB?
    Outline of Talk
    1. Brief Introduction- What’s available?
    2. General Features from the MaizeGDB homepage
    3. Searching the Database
    4. Community Curation
    5. Usage Example: Walking to genes
    6. Case Study: Find SSRs close to mac1
    7. Finding QTLs
    8. Finding cytological position from genetic position
    Outline of Talk
    1. Brief Introduction- What’s available?
    2. General Features from the MaizeGDB homepage
    3. Searching the Database
    4. Community Curation
    5. Usage Example: Walking to genes
    6. Case Study: Find SSRs close to mac1
    7. Finding QTLs
    8. Finding cytological position from genetic position
    Walking to a Gene for non-geneticists

    What is it? A way to identify the sequence of a gene of interest


    using genetic and sequence information

    Why would you do it? If you have a cool mutant that you
    know is heritable and non-allelic to other (cloned) mutants,
    you would want to know the sequence of the gene in order to
    understand more about the normal function of the gene.

    How do you walk? Genetically map your mutation, and


    Progressively narrow the region in which the gene is located,
    until you are down to sequence.
    Walking to gene is just high resolution
    genetic mapping
    Left

    Genetic map
    mutant
    Walking to gene is just high resolution
    genetic mapping Right
    Left

    Genetic map
    mutant
    Walking to gene is just high resolution
    genetic mapping Right
    Left

    Genetic map
    mutant
    Walking to gene is just high resolution
    genetic mappingRight
    Left

    Genetic map
    mutant
    Walking to gene is just high resolution
    genetic mappingRight
    Left

    Genetic map
    mutant
    Walking to gene is just high resolution
    genetic mapping
    Left Right

    Genetic map
    mutant
    Walking to gene is just high resolution
    genetic mapping
    Left Right

    Genetic map
    mutant
    Walking to gene is just high resolution
    genetic mapping
    Left Right

    Genetic map
    PCO068796
    umc1504 mutant

    Sequence umc1504
    PCO068796
    An Overview of How To Walk to a Gene
    1. Make a Mapping Population (500-1000 progeny).
    2. Map your mutant to a bin.
    -Bulk segregant analysis or standard mapping
    3. Use MaizeGDB to find markers in that bin that are likely to
    work with the inbreds in your mapping population.
    4. Use the markers to do fine structure mapping (using the
    same mapping populations) then relate your genetic
    position to the BAC map (currently requires other
    databases).
    5. Once you are down to a reasonable number of genes on
    one-three BACs, use RT-PCR or sequence to find
    differences in wt vs. mutant.
    An Overview of How To Walk to a Gene
    1. Make a Mapping Population.

    xyz(B73) x + (Mo17)
    xyz(B73) + (Mo17)
    (Introgressed into B73)

    + (Mo17) x xyz(W23)
    xyz(B73) xyz(W23)

    Mapping population: B73 alleles with the xyz gene


    Aim to get 500-1000 progeny in you mapping population
    An Overview of How To Walk to a Gene
    1. Make a Mapping Population.

    + (Mo17) x xyz(W23)
    xyz(B73) xyz(W23)

    + (Mo17) and xyz(B73) 1:1


    xyz(W23) xyz(W23)
    Aim to get 500-1000 progeny in you mapping population
    An Overview of How To Walk to a Gene
    1. Make a Mapping Population.
    2. Map your mutant to a bin.
    -Bulk segregant analysis or standard mapping
    SSR: simple sequence repeat
    (length polymorphism)
    PCR Primer
    (CA)n (CA)m

    variation between strains in number of repeats


    at a given locus

    PCR yields products of different size:


    An Overview of How To Walk to a Gene
    1. Make a Mapping Population.
    2. Map your mutant to a bin.
    -Bulk segregant analysis or standard mapping

    Lets say your mutant mapped to bin 3.04


    An Overview of How To Walk to a Gene
    1. Make a Mapping Population.
    2. Map your mutant to a bin.
    -Bulk segregant analysis or standard mapping
    3. Use MaizeGDB to find markers in that bin that are likely to
    work with the inbreds in your mapping population.
    An Overview of How To Walk to a Gene
    1. Make a Mapping Population.
    2. Map your mutant to a bin.
    -Bulk segregant analysis or standard mapping
    3. Use MaizeGDB to find markers in that bin that are likely to
    work with the inbreds in your mapping population.

    Heres another way to get to close SSRs


    An Overview of How To Walk to a Gene
    1. Make a Mapping Population.
    2. Map your mutant to a bin.
    -Bulk segregant analysis or standard mapping
    3. Use MaizeGDB to find markers in that bin that are likely to
    work with the inbreds in your mapping population.
    4. Use the markers to do fine structure mapping (using the
    same mapping populations) then relate your genetic
    position to the BAC map (currently requires other
    databases).
    Lets say that your mutant is between two markers:
    umc1504 and PCO068796
    (genetically right next too each other).

    Go to MaizeSequence.org to find the BAC sequences


    An Overview of How To Walk to a Gene
    1. Make a Mapping Population.
    2. Map your mutant to a bin.
    -Bulk segregant analysis or standard mapping
    3. Use MaizeGDB to find markers in that bin that are likely to
    work with the inbreds in your mapping population.
    4. Use the markers to do fine structure mapping (using the
    same mapping populations) then relate your genetic
    position to the BAC map (currently requires other
    databases).
    5. Once you are down to a reasonable number of genes on
    one-three BACs, use RT-PCR or sequence to find
    differences in wt vs. mutant.
    Outline of Talk
    1. Brief Introduction- What’s available?
    2. General Features from the MaizeGDB homepage
    3. Searching the Database
    4. Community Curation
    5. Usage Example: Walking to genes
    6. Case Study: Find SSRs close to mac1
    7. Finding QTLs
    8. Finding cytological position from genetic position
    Outline of Talk
    1. Brief Introduction- What’s available?
    2. General Features from the MaizeGDB homepage
    3. Searching the Database
    4. Community Curation
    5. Usage Example: Walking to genes
    6. Case Study: Find SSRs close to mac1
    7. Finding QTLs
    8. Finding cytological position from genetic position
    Genetic vs Cytolgical MAPs
    Outline of Talk
    1. Brief Introduction- What’s available?
    2. General Features from the MaizeGDB homepage
    3. Searching the Database
    4. Community Curation
    5. Usage Example: Walking to genes
    6. Case Study: Find SSRs close to mac1
    7. Finding QTLs
    8. Finding cytological position from genetic position

    Open Discussion
    THANK YOU!!

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