Maize Genetics, Genomics, Bioinformatics workshop

1. Maize Genetics, Genomics, Bioinformatics workshop Doreen Ware ARS USDA Cold Spring Harbor Laboratory

2. The Plan • Acknowledgements • Maize Sequences • Massaging of data sets • Maize FPC, MMP unigene alignments • Comparative map views • Pair wise alignments of maize genetic map t to the rice pseudomolecule, Gramene CMap views • How might this be useful?

3. CSHL Lincoln Stein Wei Zhao Kiran Ratnapu Ken Clark ? Lenny Teytelman ?

4. Cornell Susan McCouch Noel Yap

5. Maize sequences Red genomic, Blue expressed, Green clustered, Black genetic map Pink FPC

6. ESTs (Expressed Sequence Tag) • Clusters of ESTs • Tug- Plantgdb (www.plantgdb.org) • GI-TIGR (www.tigr.org) • Unigenes (Maizegdb) private sequences included deposited in Genbank • NCBI has unigenes

7. Genomic Sequence • Complete BACs • Genomic Survey Sequences (GSS) • BAC ends • 100 skim bacs • Methyl filtered • Hi-Cot filtered

8. Rice and Maize Synteny Analysis public data sets • Maize Mapping Project (MMP) • AGI Maize FPC map Cone et al., 2002 • www.maizemap.org, www.genome.arizona.edu/FPC/maize/ • IBM 2 neighbors Map • www.maizegdb.org • International Rice Sequencing Project (IRGSP) • Rice Genome Sequence • rgp.dna.affrc.go.jp/IRGSP/ • TIGR rice assembly • www.tigr.org

9. Leverage synteny of cereal genomes in the absences of complete sequence • Rice sequenced based maps • Cereal genetic and FPC maps • Establish common anchor points between the genetic and physical maps • Extend information available from the genetic maps of each species to the physical maps (leverage work of genetic systems) • Quantitative Trait Loci (QTL) and Mutants

10. Maize FPC map • Identify features to use • Maize unigene overgos (MMP unigenes) • Maize genetic markers • Identify high confidence features for correspondence to rice. • Assign a position for the feature on a contig

11. Finger Print Contig Maps • FPC maps consist of contigs of clustered BACs • The BACs are represented by imaged bands • Bands represent restriction enzyme digest fragments of the BAC clones • cccG/ATATCcccgga…ccggatcaG/ATATCacc • Features are anchored to BACs in the contigs

12. What is an overgo? • ~42 bp oligo sequence used for hybridization • Derived from the MMP unigenes • Gene specific?

13. BACs overgos MMP overgo positions on the maize FPC map contig

14. How many MMP overgo positions are on the maize FPC map? • All MMP Unigene overgo positions • 15,574 contig Contig B Contig A overgos 4 overgo positions on two contigs using 3 overgo probes

15. How many FPC contigs does an overgo hit?

17. How many BACs does an overgo hit in a contig?

19. Select overgos with at least two BACs hybridized in an FPC contig. • To remove potential false positives, require an overgo be found on more than one BAC in a Maize FPC contigs. • Red and pink are accepted blue is rejected • 8864 contig BACs overgos

20. Generate a position for the overgo on the FPC contig • To establish a rough order of the overgo on the contig the position of the BACs the overgo hit in the FPC contig is used. This establishes a relative order of the overgos within the contig • Maximum start position of a BAC • Minimum end position of a BAC contig BACs overgos

21. Align MMP clusters to the rice genome • Sequenced based alignments of features to the rice genome using BLAT. • Single best match in the rice genome • 6,771 maize unigenes (63%)

22. Filter based upon match-length Matches with less than 150bp match-length were removed leaving 7,770 hits.

23. Distribution of percent identity of Maize Cornsensus unigenes mapped to rice The hits represent 6,692 unique Clusters (62% of the total 10,678).

24. What is the distribution of the maize unigenes across the rice genome?

25. Calculate adjacent distances between pairs of unigenes on maize contigs and their distance of the rice genome Maize Rice

26. 50Kb_1MB Colinear gene pairs 400,000 bps or lower

27. 90% of colinear maize overgos contig-pairs fall under 400,000 bps are found within 165,000 bps on rice

28. A maize contig span will be defined as syntenic if it contains a unigenes pair that is less than 400 kb apart on the rice genome Maize Rice

29. Maize Contig 417 CTG 417 Anchored to the maize chromosome 8 by marker UMC1905

30. Plot the colinear rice and maize spans that contain genetic marker information • What regions of the rice genome are syntenic with maize? • What regions of the rice genome have no synteny with maize? • What regions of the maize genome have no synteny to rice?

31. Maize-Rice Colinear intervals with anchored maize genetic position

32. Genetic loci with sequence mapped to rice genome

33. Blue - Genetic markersRed -Common Green- Colinear FPC contigs

34. What can rice do for maize? • Where colinear regions exist to rice • Provide potential genetic neighborhood of unanchored maize contigs • Provide link to other cereal genetic and physical maps • Provisional order of cereals features where no orientation is known. (Wheat deletion bin map, Maize filtered reads?) • Candidate sequence for marker screening development • Where limited or no colinear regions exist to rice • Rethink candidate gene approach?

35. Provide potential genetic neighborhood of unanchored maize contigs chromosome 8 (red),3 (blue)and unknown (black)

36. Comparative maps with other cereals • Wheat EST deletion map (new) • Sorghum RFLP map from Paterson (new) • Rice TIGR assembly (new) • Maize curated FPC

37. Rice chromosome 1 to Wheat deletion map, Sorghum genetic map, and Maize FPC map

38. Provisional order of cereals features where no orientation is known.

39. Summary The maize physical map can provide a provisional order for the maize sequences that have been anchored. In syntenic regions the rice sequence can serve as an anchor to define contact points between cereal genomes In syntenic regions the rice sequence can provide a provisional order to cereal sequences