Sequencing of Medicago truncatula genome and studies of metabolic pathways

1. Sequencing of Medicago truncatula genomeand studies of metabolic pathways Iryna Sanders University of Oklahoma Roe’s Lab

2. Why Study Legumes • Second after grasses economically important agronomical family • Rich in protein - 33% of human dietary consumption • Alternative fuel • Fix nitrogen via symbiosis with Rhizobium bacteria • Natural fertilizer • Rich source of natural health benefiting product (e.g. flavonoids and isoflavonoids) • Important ecosystem component as they can occupy lands unsuitable for other plants Why Medicago truncatula is a Model Legume • Small diploid genome (470 Mbp) • Self-fertilized • Short seed-to-seed generation time • High transformation efficiency • Large collection of phenotypic mutants and naturally occurring ecotypes

3. Sequencing Methods Fluorescent-dye terminator Massively parallel pyrosequencing ABI-3730 Roche-454 Randomly sheared DNA pUC library construction E.coli transformation Subclone isolation Sequencing using fluorescently labelled ddNTPs Randomly sheared DNA DNA library construction Immobilization on magnetic beads Emulsion PCR Deposit bead library onto the PicoTiterPlate Sequencing based on synthesis using DNA polymerase, ATP sulfurylase, luciferase and apyrase

4. Results obtained from a combination of two sequencing methods Fluorescent-labeled dye terminator sequencing on the ABI 3730 alone Fluorescent-labeled dye terminator method combined with pyrosequencing on the Roche-454 $ 10,000 $ 2,000 36 contigs done

5. Data Analysis and Annotation Schema Sequence Screen for Repeats BLASTN (rRNA) tRNA-ScanSE (tRNA) FGeneSH and GenScan (Gene prediction) Metabolic Reconstruction using the KEGG database Comparative Analysis: * PIP-maker - Local Alignment * ClustalW - Gaped alignment * Vista - Global alignment

6. Comparison of the 8 Medico truncatula chromosomes

7. information storage and processing cellular processes and signaling metabolism poorly characterized no metabolic hits no hits KOG results for predicted proteins of Medicago truncatula chromosomes 0 1 2 4 3 5 8 6 7

8. TCA Other carbohydrate metabolism Energy metabolism Lipid metabolism Nucleotide metabolism Aromatic amino acid metabolism Other amino acid metabolism Glycan biosynthesis/ metabolism Terpenoid metabolism Flavonoid metabolism Cofactor and vitamin metabolism KEGG results for predicted metabolism proteins for Medicago truncatula chromosomes 2 0 1 5 3 4 8 6 7

9. metabolism primary secondary not directly function in the processes of growth and development keep the cell alive normal anabolic and catabolic processes resulting in assimilation, respiration, transport, and differentiation produced in all cell produced in specialized cells sugars, amino acids, nucleotides antibiotics, plant defenses chemicals Metabolism

10. Phylogenetic tree for the tricarboxylic cycle enzymes (TCA) Among 8 enzymes of TCA cycle: 3 enzymes have 2 or more copies Among duplicated copies there are those that share the highest identity and members that diverged from the main group Duplicated genes occur as single duplication in the genome AC – aconitase, IDH – isocitrate dehydrogenase, KGDH – α ketoglutarate dehydrogenase complex, SS – succinyl CoA synthetase, SDH – succinate dehydrogenase, MD – malate dehydrogenase, CHS – chalcone synthase (negative control

11. Phylogenetic tree for the enzymes of aromatic amino acid biosynthesis Among all enzymes of this pathway: only 4 enzymes have 2 or more copies prephenate dehydratase duplicates share 100% protein identity other duplicated genes share 70% or less identity Duplicated genes occur as single duplication in the genome APT-anthranilate-phosphoribosyl transferase, CM-chorismate mutase, PD-prephenate dehydratase PPD-Prephenate dehydrogenase, 3DD/S5D-3-dehydroquinate dehydratase/shikimate5-dehydrogenase, 5ESPS-5-enolpyruvylshikimate 3-phosphate synthase, PAI-phosphoribosylanthranilate isomerase, AS-anthranilate synthase, CHS – chalcone synthase (as a negative control).

12. Flavonoid biosynthesis enzymes % protein identity if applicable # of copies in the Medicago genome Branch Point enzyme? Type of duplication Name of gene L-phenylalanine ammonia-lyase no 89 single 5 cinnamic acid 4-hydroxylase no 1 single 4-coumarate:coenzymeA ligase no 3 16 single chalcone synthase yes 20 76 93 86 single, tandem, cluster chalcone reductase no 6 72.5 single isoflavone 4’-O-methyltransferase no 7 58.3 single. isoflavone reductase no 5 48.3 single, 32 tandem vestinone reductase no 7 61.5 single dihydroflavonol reductase no 6 51 single, 68 tandem leucoanthocyanin reductase no 1 single flavonoid glucosyl transferase no 5 34.3 single leucoanthocyanidin dioxygenase no 1 single flavonol synthase no 37.8 single, 10 45 tandem flavone-3-Hydroxylase no 4 59.5 single

13. Comparison of genome organization of primary metabolism genes vs. secondary metabolism genes primary secondary • Small portion of genes are duplicated • Type of duplication – single • Average protein identity for duplicated genes is approximately the same as the secondary metabolism genes • There are members of one protein family that share high percentage of identity while others differ greatly • Branchpoint gene(s) do not have more copies than other members • Vast majority of genes are duplicated • Type of duplication – single, tandem, and cluster • Single duplicates have the lowest level of protein identity and tandem duplication have the highest, while members of clusters are in between • There are no duplicates that share near 100% of identity • Branchpoint gene(s) have a very large number of copies comparing with non-branchpoint genes

14. Primary and secondary metabolic pathway average gene expression changes in M. truncatula after innoculation with Rhizobium comparing to uninfected roots Aromatic amino acid TCA Flavonoid Triterpenoid

15. All flavonoid gene expression

16. ConclusionsThis slide needs work!! It is more of a summary slide and not a conclusion slide. A conclusion has two parts. We observed something and what that observation leads us to conclude that something new is proposed or thought of. In other words WHAT THAT OBSERVATION MEANS is some new thing. Please correct this slide. • Except chromosomes 5 and 6, the Medicago truncatula chromosome arms have high percentage of genes and low percentage of repeated sequences • The largest groups of genes encode genes of carbohydrate, energy, and amino acid metabolism for protein production characteristic for a legume and to ensure legume prosperity in a carbohydrate world • Different types of metabolic genes are spread unevenly among all Medicago chromosomes, presumably to ensure the plasticity of a plant • Primary metabolic genes are less duplicated that genes of secondary metabolism because they express continuesly and at high rate • Secondary metabolism duplicated genes occur in various types of duplications and have much more copies that genes of primary metabolism because they are expressed as a response to different environmental condition (stresses) • There is a connection between expression of aromatic amino acid biosynthesis genes and the flavonoid and triterpenoid biosynthesis genes after inoculation with a symbiotic Rhizobia • Although the average expression for flavonoid genes could be up regulated, the expressions of duplicated genes can show a significant difference to give an adequate response to environmental stress

17. Acknowledgements • Dr. Bruce A. Roe • Dr. F. Najar, Hongshing Lau, Steve Kenton • Shweta Deshpande, Majesta O’Bleness, Jing Yi, Leo Sukharnikov • Dr. Yuohong Tang • Noble Foundation • NSF • All members of Dr. Roe’s lab • Graduate Committee members: Dr. A. West, Dr. P. Klebba, Dr. Richter-Addo, Dr. Conway • Mattew from Dr. Conway’s lab