1. Genetic and Genomic Resources in Maize Georgia Davis Asst. Professor Div. Plant Sciences davisge@missouri.edu

2. Overview Genetic map Physical map Anchoring via Molecular Markers

3. What is Genomics? Genomics science that combines genetics, high through-put robotics and bioinformatics to derive large amounts of high quality biological information that will allow a greater understanding of the organism being studied.

4. Structural Genomics zScience related to discovery of gene order and organization on a chromosome. yGenetic mapping yPhysical mapping yIn situ hybridization

5. Functional Genomics zScience related to the discovery and definition of the function of genes Whole plantCellular Biochemical RNA C16 C18 C20 C22 C24 C26 AldehydesAlcohols Wax Esters

6. The Maize Genome in Perspective zMaize2500 MB zHuman2500 MB zSoybean1500 MB zSorghum 900 MB zTomato 950 MB zRice 450 MB zBacteria 5 MB

7. How maize compares to other species in size zHumanequal zSoybean 1.7X zSorghum 2.7X zTomato 2.6X zRice 5.5X zBacteria 500X

8. Maize Genetic Resources zb7 d10 gl2 al1 kn1

9. Maize Genetic Resources orp1 mn1 tu1 a1 su1 y1 pr1 sh1 wx1 b1 pl1 vp1 te1

10. Maize Genetic Resources z1685 mutants with images in MDB z>5000 mutants available z>10,000 alleles in MDB www.maizegdb.org

11. Maize Genetic Resources z49 QTL experiments in MDB z59 genetic maps in MDB z7850 mapped loci in MDB

12. Mapping Maize at UMC zGenetic mapping is based on DNA fingerprint data of offspring from two parents which differ in their appearance. ySimilar fingerprint data for two gene indicates they are physically close together on a chromosome. zPhysical mapping is a laboratory based approach and involves identifying small overlapping DNA fragment to reconstruct whole maize chromosomes.

13. Molecular mapping Digest DNA Electrophorese - + Southern blot Hybridize with probe

14. Scoring data A B H H A B A H

15. Recombination and Mapping z Assume that the frequency of crossing-over is equal along the chromosome. z Two genes that are very close to one another will have a lower likelihood of having a cross-over between them than two genes that are very far apart.

16. Recombination and Mapping z So, we can determine the relative distance between genes by counting the number of recombinant genotypes for each pair of genes.  Lots of recombinants = far apart  Few recombinants = close together

17. BHBBAHBHHBHHBHB umc157 HHBBABBHHBBBBAB umc76 BHBBABHAHHBHBAB asg45 BHBBABBAHHBHBAB zb4 BHBBHBHAHHBHBAB csu3 How Maximum Likelihood Works

18. BHBBAHBHHBHHBHB umc157 BHBBABHAHHBHBAB asg45 HHBBABBHHBBBBAB umc76 BHBBABBAHHBHBAB zb4 BHBBHBHAHHBHBAB csu3

19. ESTs Defined zEST is an acronym for Expressed Sequenced Tags. zcDNAs with map locations. zESTs represent the genes active in a particular tissue at specific stage of development. zThe cellular function of an EST can often be derived by comparison to sequence databases.

20. zESTs are important markers for physical mapping. zEST mapping, coupled with trait mapping, will help link genes to phenotypes. zESTs are the basic building blocks for high density “gene chips” EST Mapping: Utility

21. Efficient Markers for Maize Simple Sequence Repeats(SSRs) zDevelop SSR markers zMap SSRs genetically zUse SSRs to anchor physical map to genetic map zUse SSRs to map QTLs more efficiently

22. SSRs: What and how? zDinucleotide and trinucleotide repeats. ex. CACACACACA zDifferent corn lines have different numbers of repeats. (CA) 10, (CA) 14 zThese differences can be seen on a high resolution agarose gel. zCan be used for mapping.

23. SSR Mapping C A T G

24. Trait Mapping zLinks phenotypes to genetic locations zPrecursor to linking phenotypes to genes zProvides tools for functional analysis of gene expression

25. Aspergillus flavus Infection via silk channel wounds in kernels cracks in kernels

26. Aflatoxin Aflatoxin B1  Potent carcinogen.  Associated with liver cancer in humans.

27. Molecular mapping z Compare the DNA fingerprint with grain toxin levels z Look for bands on fingerprint associated with high toxin and those associated with low toxin DNA fingerprint Gene 1 Mp313E Va35 123456 LoHiInt.Lo HiInt. LoHiInt.Lo HiInt. Gene 2

28. Mapping Maize at UMC zGenetic mapping is based on DNA fingerprint data of offspring from two parents which differ in their appearance. ySimilar fingerprint data for two gene indicates they are physically close together on a chromosome. zPhysical mapping is a laboratory based approach and involves identifying small overlapping DNA fragment to reconstruct whole maize chromosomes.

29. Mapping Maize at UMC Physical Mapping zLarge fragments are cloned into Bacterial Artificial Chromosomes (BACs). zDNA fragments can easily be retrieved for gene hunting experiments. zA complete physical map is needed before DNA sequencing of the chromosomes can begin.

30. Maize HindIII BAC library zMade at CUGI zAverage insert size is 135 kb. zTotal coverage is 14X. zFingerprinting and anchoring of 5X underway.

31. Physical Map Constructed in the laboratory by assembling overlapping DNA fragments. Each fragment can be easily retrieved for experiments which can localize genes of interest. DNA fragments cloned into Bacterial Artificial Chromosomes (BACs) Chromosome 1

32. Integrating the Genetic and Physical Maps to Form a Single Map zMolecular markers serve as anchor points for the two maps since they can be mapped both genetically and physically zTypes of Molecular Markers yRFLPs ySimple Sequence Repeats (SSRs) yExpressed Sequence Tags (ESTs) yMarkers from other species

33. Maize Physical Mapping zWhole-genome characterization of HindIII in progress. zAnchor with AFLPs and low copy RFLPs zConsiderations: yRepetitive elements ySyntenic duplicated regions B73 Maize BAC library probed by asg20 single copy clone

34. Maize Physical Mapping CentA-LTR sequence bnl6.16 - Single copy RFLP

35. Integrated Genetic and Physical Map Plant Height Disease Resistance Kernel Color Tassel Branching Drought Resistance Unknown Function (EST) Chromosome 1 GeneticPhysical

36. Synteny in Crop Plants zThe order of genes along chromosomes is conserved in many crop plants. yMaize-Sorghum-Rice yTomato-Pepper-Potato zThis is known as synteny and occurs in animals as well. yMouse-Rat-Humans zThe Missouri Maize Project is working to characterize maize-sorghum synteny.

37. Maize Synteny With Close Relatives The Missouri Maize Project is working to characterize maize-sorghum-rice synteny. This will allow desirable plant traits to be transferred across species. SorghumMaizeRice

38. Informatics Tasks What good is the data if you can’t access it? zProject data curation zCommunity curation via MaizeDB zDissemination from MaizeDB

39. Excel Spreadsheets Project Data Curation LabDB MaizeDB Scripts Scripts

40. What do we use the tools for? zIdentifying genes for important agronomic traits. yInsect resistance yDisease resistance yDrought tolerance yCold/heat tolerance yGrain quality