Comprehensive Genetic, Physical, and Database Resources for Maize

1. Comprehensive Genetic, Physical, and Database Resources for Maize

2. University of Missouri and USDA-ARS Ed Coe Mike McMullen Jack Gardiner Mary Polacco Karen Cone Mannie Liscum Su-Shing Chen Georgia Davis Clemson University Rod Wing Ralph Dean University of Georgia Andrew Paterson

3. Objectives • Integrated physical and genetic map resource • Efficient, high-resolution genetic mapping resources • Enhanced data curation, access, and representation

4. Overview Genetic map Anchoring via Molecular Markers Physical map INFORMATICS

5. 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.

6. Structural Genomics • Science related to discovery of gene order and organization on a chromosome. • Genetic mapping • Physical mapping • In situ hybridization

7. Functional Genomics • Science related to the discovery and definition of the function of genes C16 C18 C20 C22 C24 C26 Aldehydes Alcohols Wax Esters RNA Whole plant Cellular Biochemical

8. The Maize Genome in Perspective • Maize 2500 MB • Human 2500 MB • Soybean 1500 MB • Sorghum 900 MB • Tomato 950 MB • Rice 450 MB • Bacteria 5 MB

9. How maize compares to other species in size • Human equal • Soybean 1.7X • Sorghum 2.7X • Tomato 2.6X • Rice 5.5X • Bacteria 500X

10. Maize Genetic Resources al1 d10 gl2 zb7 kn1

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

12. Maize Genetic Resources • 1685 mutants with images in MDB • >5000 mutants available • >10,000 alleles in MDB www.agron.missouri.edu May 8, 2000

13. Maize Genetic Resources • 49 QTL experiments in MDB • 59 genetic maps in MDB • 7850 mapped loci in MDB May 8, 2000

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

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

16. Scoring data A B H H A B A H

17. Recombination and Mapping • Assume that the frequency of crossing-over is equal along the chromosome. • 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.

18. Recombination and Mapping • 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

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

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

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

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

23. Efficient Markers for MaizeSimple Sequence Repeats(SSRs) • Develop SSR markers • Map SSRs genetically • Use SSRs to anchor physical map to genetic map • Use SSRs to map QTLs more efficiently

24. SSRs: What and how? • Dinucleotide and trinucleotide repeats. ex. CACACACACA • Different corn lines have different numbers of repeats. (CA)10, (CA)14 • These differences can be seen on a high resolution agarose gel. • Can be used for mapping.

25. SSR Mapping CA T G

26. Trait Mapping • Links phenotypes to genetic locations • Precursor to linking phenotypes to genes • Provides tools for functional analysis of gene expression

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

28. Aflatoxin Aflatoxin B1 • Potent carcinogen. • Associated with liver cancer in humans.

29. Gene 1 Mp313E Va35 1 2 3 4 5 6 Lo Hi Int. Hi Lo Int. Lo Int. Gene 2 Lo Hi Int. Hi Lo Int. Lo Int. Molecular mapping • Compare the DNA fingerprint with grain toxin levels • Look for bands on fingerprint associated with high toxin and those associated with low toxin DNA fingerprint

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

31. Mapping Maize at UMCPhysical Mapping • Large fragments are cloned into Bacterial Artificial Chromosomes (BACs). • DNA fragments can easily be retrieved for gene hunting experiments. • A complete physical map is needed before DNA sequencing of the chromosomes can begin.

32. Maize HindIII BAC library • Made at CUGI • Average insert size is 135 kb. • Total coverage is 14X. • Fingerprinting and anchoring of 5X underway.

33. 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

34. Integrating the Genetic and Physical Maps to Form a Single Map • Molecular markers serve as anchor points for the two maps since they can be mapped both genetically and physically • Types of Molecular Markers • RFLPs • Simple Sequence Repeats (SSRs) • Expressed Sequence Tags (ESTs) • Markers from other species

35. Maize Physical Mapping • Whole-genome characterization of HindIII in progress. • Anchor with AFLPs and low copy RFLPs • Considerations: • Repetitive elements • Syntenic duplicatedregions B73 Maize BAC library probed by asg20 single copy clone

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

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

38. Synteny in Crop Plants • The order of genes along chromosomes is conserved in many crop plants. • Maize-Sorghum-Rice • Tomato-Pepper-Potato • This is known as synteny and occurs in animals as well. • Mouse-Rat-Humans • The Missouri Maize Project is working to characterize maize-sorghum synteny.

39. 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. Maize Sorghum Rice

40. Informatics Tasks What good is the data if you can’t access it? • Project data curation • Community curation via MaizeDB • Dissemination from MaizeDB

41. Project Data Curation Excel Spreadsheets Scripts LabDB Scripts MaizeDB

42. What do we use the tools for? • Identifying genes for important agronomic traits. • Insect resistance • Disease resistance • Drought tolerance • Cold/heat tolerance • Grain quality