The Oryza Map Alignment Project (OMAP) and its utility for rice breeding and genetics

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arizona.edu Rice sequence is done What s Next?

mutant analysis transposon tagging, tilling Proteomics & metabolomics advanced

1 The Oryza Map Alignment Project (OMAP) and its utility for rice breeding and genetics David Kudrna Arizona Genomics Institute University of Arizona Tucson, AZ Rice sequence is done What s Next? Complete functional characterization of the rice genome gene and whole genome arrays mutant analysis transposon tagging, tilling Proteomics & metabolomics advanced molecular breeding strategies for cultivar development to feed the world comparative genome studies (between the cereals and within Oryza) 1

2 Phylogenetic AA BB { O. sativa; O. nivara; O. rufipogon; O. glaberrima O. punctata Tree of BBCC O. minuta Oryza CC O. officinalis CCDD O. alta EE O. australiensis KK HHKK O. coarctata FF O. brachyantha HH HHJJ O. ridleyi JJ GG O. granulata Evolutionary relationships of the rice genomes; (Ge et al., PNAS-96: ,1999) Dashed lines indicate origins of allotetraploids; indicate maternal parents; indicate unidentified diploid genomes O. minuta O. granulata O. rufipogon O. sativa O. alta O. coarctata O. ridleyi O. officinalis Wild species: phenotypically inferior grass like plants, but a valuable genetic resource D. Brar (IRRI) 2

3 Genome composition, ploidy level and distribution of Oryza species Species Genome 2n Distribution (A) O. sativa complex O. sativa AA 24 world wide O. nivara AA 24 tropics O. rufipogon AA 24 tropics & subtropics O. glaberrima AA 24 West Africa O. barthii AA 24 Africa O. longistaminata AA 24 Africa O. meridionalis AA 24 Tropical Australia O. glumaepatula AA 24 South & Central America Species Genome 2n Distribution (B) O. officinalis complex O. punctata BB, BBCC 24, 48 Africa O. officinalis CC 24 Tropical and subtropical Asia O. rhizomatis CC 24 Sri Lanka O. eichingeri CC 24 South Asia and East Africa O. minuta BBCC 48 Philippines and Papua New Guinea O. latifolia CCDD 48 South & Central America O. alta CCDD 48 South & Central America O. grandiglumis CCDD 48 South & Central America O. australiensis EE 24 Tropical Australia 3

4 Species Genome 2n Distribution (C) O. meyeriana complex O. granulata O. meyeriana GG GG Southeast Asia South & Southeast Asia (D) O. ridleyi complex O. ridleyi HHJJ 48 Southeast Asia O. longiglumis (E) Out group complex HHJJ 48 Indonesia, Papua New Guinea O. brachyantha FF 24 Africa O. schlechteri HHKK 48 New Guinea O. coarctata HHKK 48 Eastern India Some wild species of Oryza and their useful traits SPECIES GENOME USEFUL TRAITS O. sativa O. glaberrima O. longistaminata O. rufipogon O. minuta O. officinalis O. latifolia O. australiensis O. brachyantha O. granulata O. ridleyi O. coarctata AA AA AA AA BBCC CC CCDD EE FF GG HHJJ HHKK Cultivated worldwide Cultivated in West Africa; increased tolerance to biotic and abiotic stresses; weed competitiveness, drought tolerance Resistance to BB, stemborer; drought avoidance Resistance to BB, sheathblight, abiotic stresses, CMS, high iron and zinc (55 mg vs 22) Resistance to BPH, BB, blast, sheath blight Resistance to BPH, BB, GLH Resistance to BPH, high biomass, high iron and calcium (240 mg vs 78) Resistance to BPH, drought avoidance Resistance to BB, stemborer Shade tolerance, adaptation to aerobic soil conditions Resistance to BB, blast, stemborer Salt tolerance BB=bacterial blight; BPH=brown plant hopper; GLH=green leaf hopper; CMS=cyto male sterility 4

Development Genome Organization Polyploidy Domestication Comparative Genomics Gene Regulatory Networks

5 Oryza Map Alignment Project (OMAP) Project Overview Long-term objective: To create a genome-level closed experimental system for the genus Oryza that can be used as a research platform to study: Evolution Development Genome Organization Polyploidy Domestication Comparative Genomics Gene Regulatory Networks Positional Cloning Crop Improvement Oryza Map Alignment Project - Team AGI Purdue OMAP CSHL AGCoL OMAP Advisory Committee Meetings 5

6 Immediate Objectives 1. Construct 12 High-quality BAC Libraries from Representatives of the 10 Genome Types 2. Generate 12 Fingerprint/End Sequence Maps for the wild Oryza genomes 3. Global Alignment of the 12 Maps to Reference O. sativa genome 4. Detailed Reconstruction of Chromosomes 1, 3 & Data Mining (e.g. SNPs, Repeat Analysis, Structural Variation) 6. Data Presentation (e.g. WebFPC, SyMAP, Gramene) 6

What is a BAC clone?? A transformed E. coli bacterium that contains a large fragment (100-200kbp) of a genome that is cloned into a plasmid vector. Bacterial Artificial Chromosome E.

coli w/ cloned plasmid) Grow and make millions of copies BAC Library Construction BamHI Sph I Hind III HMW DNA (nuclei embedded in agarose) LoxP Not I Not I lacz Sac I Sal I parb para cos N

7 What is a BAC clone?? A transformed E. coli bacterium that contains a large fragment ( kbp) of a genome that is cloned into a plasmid vector. Bacterial Artificial Chromosome E. coli cell genomic DNA fragment ~140 kbp plasmid ~7 kbp ligation transformation cloned plasmid circular double stranded DNA BAC clone (E. coli w/ cloned plasmid) Grow and make millions of copies BAC Library Construction BamHI Sph I Hind III HMW DNA (nuclei embedded in agarose) LoxP Not I Not I lacz Sac I Sal I parb para cos N pbelobac11 (7.5 kb) Sac I Sal I CM r OriS repe Enzyme digested, dephosphorylated vector Partial digested, size selected insert DNA Ligation, transformation into E.coli via electroporation Coverage of genome size of inserts x #clones Pick recombinant clones and store at -80C in 384 well plates BAC library-- Individual clones are arraryed into plates. 7

8 Oryza Map Alignment Project (OMAP) BAC Libraries Oryza Species Genome designation Final Genome Size IRGC Plant Accession No. Accession Origin AGI BAC library name Whole lib.: # Clones O. glaberrima AA 357Mbp Africa OG_BBa O. nivara AA India OR_BBa O. rufipogon AA Malaysia OR_CBa O. punctata BB Africa OP Ba O. officinalis CC Thailand OO Ba O. minuta BBCC Philippines OM Ba O. alta CCDD So. America OA_BBa O. australiensis EE Australia OA_ABa O. brachyantha FF Africa OB Ba O. granulata GG Thailand OG_ABa O. ridleyi HHJJ Thailand OR_ABa O. coarctata HHKK Bangladesh OC Ba BAC libraries, high density filters can be ordered from the AGI BAC/EST Resource Center ( Ammiraju et al (2006) Genome Research 16(1): What is a BAC end sequence (BES)?? ( sometimes called STC) The DNA terminal sequence of a BAC clone initiated from the cloning vector and reading into the insert; both forward and reverse directions. Grow and make millions of cells that contain plasmid DNA BAC clone Extract plasmid DNA Perform sequencing reaction and analyze ~700bp..AATGCCGTAATGTCCGGG M13 rev primer AATGCCGTAATGTCCGGG.. ~700bp T7 fwd primer genomic rice DNA plasmid genomic rice DNA 8

Data Release http://www.omap.org/status.

Genomics (2003) 82:378-389 End label with fluorescent ddntps

using genemapper software RE RS ddntp FD Color (Restriction

G AATTC G GATCC T CTAGA C TCGAG GG CC A G C T None

9 Data Release BAC DNA SNaPshot Fingerprinting RE digestion Luo et al, Genomics (2003) 82: End label with fluorescent ddntps Electrophoresis on capillary sequencer Size file generation using genemapper software RE RS ddntp FD Color (Restriction Endonuclease) EcoRI BamHI XbaI XhoI HaeIII (Restriction Site) G AATTC G GATCC T CTAGA C TCGAG GG CC A G C T None (Fluorescent dye label) dr6g dr110 dtamra drox (Color of restriction fragments) Green Blue Yellow Red 9

Finger Printed Contig (FPC) Software by Cari Soderlund et al, AGCoL Genome Research 10:1772-1787, 2000 Features: Tolerance -how close two

less-than-perfect matches within a contig End join merge contig end clones overlap different contigs at e value Marker addition affix

10 Finger Printed Contig (FPC) Software by Cari Soderlund et al, AGCoL Genome Research 10: , 2000 Features: Tolerance -how close two bands match Cut off probability that 2 clones overlap (as e value) Gel length number of possible band values Q-clones clones with less-than-perfect matches within a contig End join merge contig end clones overlap different contigs at e value Marker addition affix locus mark to BAC(s) for contig assembly Author and Automation friendly Band value drgb 5,000 dr110 10,000 dtamra 15,000 drox FPC Contig of O. nivara 10

O. nivara O. rufipogon O. glaberrima O. punctata O.

brachyantha Rice Physical map aligned to rice fine genetic

11 O. nivara O. rufipogon O. glaberrima O. punctata O. officinalis O. brachyantha Rice Physical map aligned to rice fine genetic map Marker cm C R C C C R Physical Maps of the AA genome species SyMAP views AA BB CC FF O. nivara 99% clones 95 % bands anchored Coverage 95 % O. rufipogon 99% clones 98 % bands anchored Coverage 98 % O. glaberrima 99% clones 99 % bands anchored Coverage 97 % 11

9 10 11 12 O. punctata 98% clones 98 % bands anchored Coverage 97 % O.

brachyantha 42% clones 90 % bands anchored Coverage 63 % Rearrangement Index

12 O. nivara O. rufipogon O. glaberrima O. punctata O. officinalis O. brachyantha Physical Maps of the BB, CC and FF genome species AA BB CC FF O. punctata 98% clones 98 % bands anchored Coverage 97 % O. officinalis 99% clones 97 % bands anchored Coverage 83 % O. brachyantha 42% clones 90 % bands anchored Coverage 63 % Rearrangement Index Computationally identify putative rearrangements between wild rice genomes and the O. sativa genome using BAC end sequences from O. rufipogon O. nivara O. glaberrima O. punctata 12

13 Rearrangement Algorithm Overview Align BES to the O. sativa pseudomolecules (IRGSP V3) 74% BES aligned Classify clones based on Paired BES alignments 89% Clones aligned 52% Paired BES for clone Detect putative Rearrangements 59% concordant pairs 41% discordant pairs Classify Rearrangements: Deletions, insertions, translocations and inversions Putative Deletion in Oryza glaberrima 217kb 222kb 192kb 227kb Expected clone size range for O. glaberrima: 91 kb 170 kb 13

14 Putative Insertion in Oryza nivara 67kb 55kb 65kb Expected clone size range for O. nivara: 108 kb 232 kb Putative Translocation in Oryza rufipogon CHR. 7 CHR. 11 OR Paired reads are expected to be on the same chromosome 14

15 Putative Inversion in Oryza glaberrima Putative Rearrangements insertions deletions inversions translocations Oryza rufipogon Oryza nivara Oryza glaberrima Oryza punctata total

Reconstruction of Chromosome 3 Wet Lab confirmation of

16 Reconstruction of Chromosome 3 Wet Lab confirmation of insertions/deletions SyMap alignment of the 3 AA genome species to Oryza sativa ssp. japonica chromosome 3 pseudomolecule. O. nivara O. rufipogon O. glaberrima Validation of BAC Clone Gel Sizing using Oryza sativa Chromosome 3, O. sativa (AA) difference Kb (insert size \ BES alignment) C 16

$nivara (AA) difference Kb (insert size \ BES alignment) Expansion Contraction C Chromosome 3$

17 Chromosome 3 Expansion and Contraction of Oryza nivara as compared to IRGSP pseudomolecule Chromosome 3, O. nivara (AA) difference Kb (insert size \ BES alignment) Expansion Contraction C Chromosome 3 Expansion and Contraction of Oryza rufipogon as compared to IRGSP pseudomolecule Chromosome 3, O. rufipogon (AA) Expansion difference Kb (insert size \ BES alignment) Contraction C 17

$glaberrima (AA) difference Kb (insert size \ BES alignment) Expansion Contraction C C The Oryza Map Alignment Project (OMAP) offers practical utility for rice breeding and genetics by: Incorporating$

18 Chromosome 3 Expansion and Contraction of Oryza glaberrima as compared to IRGSP pseudomolecule Chromosome 3, O. glaberrima (AA) Chromosome 3, O. glaberrima (AA) difference Kb (insert size \ BES alignment) Expansion Contraction C C The Oryza Map Alignment Project (OMAP) offers practical utility for rice breeding and genetics by: Incorporating wild rice alignment data into Gramene for grass comparative, syntenic and genetic analyses with free access to the public; Providing thousands of Oryza genomic sequences for marker development and marker assisted selection; Providing public access to genomic data from diverse Oryza species in the forms of FPC and SyMAP physical maps and sequence submissions to genbank; Providing public access to the genomic resources that the physical and sequence maps were derived from in the forms of BAC clones and genome screening filters for positional and QTL cloning; Providing public access to the germplasm of the OMAP species. 18

19 Objectives 1. Construct 12 High-quality BAC Libraries from Representatives of the 10 Genome Types 2. Generate 12 Fingerprint/End Sequence Maps for the wild Oryza genomes 3. Global Alignment of the 12 Maps to Reference O. sativa genome 4. Detailed Reconstruction of Chromosomes 1, 3 & Data Mining (e.g. marker development, SNPs, Repeat Analysis, positional cloning, etc.) 6. Data Presentation-- Gramene Comparative and Fine mapping (QTL, sequence maps) Polymorphic SSR Markers Identification of QTL candidate genes with allelic variation Gramene displays a powerful utilization of OMAP data for the identification of mapped markers, QTLs and diverse genetic resources for plant breeding A practical demonstration through Gramene. 19

20 FLOW of What you will see with this exercise.. QTL map from paper QTL in Gramene Rice pseudomolecule in Gramene OMAP physical map with BAC clones in Gramene Oryza rufipogon IRGC Accession #

21 OMAP data in Gramene HUNDRED Grain Weight HGRWT 21

22 C-Map Display Markers C-Map is the Gramene Comparative Mapping tool Days to Heading Germination speed Another portal into C-Map.. Grain Number Plant height Grain shattering Hundred grain wt One of the many QTL maps A different portal into C-Map 22

23 Scroll down the C-Map display to find Comparison Menu Use Gramene Annot 2006 for all rice sequence Then Redraw Map Can load on other genetic maps, etc on right side too C-Map Display of grain wt QTL aligned to rice sequence Notice SSR Marker RM104 23

gray arrows C-Map Feature types Drilled down to 40.07 40.

24 Drill down in C-Map Display of grain wt QTL aligned to rice sequence CROP down to zoom in on the region of interest use gray arrows C-Map Feature types Drilled down to Mb on Chr 1 Mouse over to see identification of a feature 24

25 CL O. rufipogon BES, OR_CBa057N10, reverse read is mapped on the IRGSP sequence Click on a feature within the C-Map display shows information about that feature O. rufipogon BAC end sequence (BES) is very near the grain wt QTL; can be used as a marker for the region Full sequence is given 25

Simple Sequence Repeat (SSR) Identification Tool Ideal for utilization of OMAP BES data for genetics and breeding Polymorphic SSRs to O. sativa in 3 AA Genomes O. nivara O. rufipogon O.

26 Simple Sequence Repeat (SSR) Identification Tool Ideal for utilization of OMAP BES data for genetics and breeding Polymorphic SSRs to O. sativa in 3 AA Genomes O. nivara O. rufipogon O. glaberrima Chr Chr Chr Chr Chr Chr Chr Chr Chr Chr Chr Chr Total

27 Mb 0 Chr Distribution of O. nivara polymorphic SSRs to O. sativa pseudomolecule (979 polymorphic SSRs) SSRs in OMAP BAC End Sequences May 10, 2006 type ploidy genome di tri tetra penta hexa Total G-size(Mb) # of SSR/Mb AA 2 pseu AA 2 indi AA 2 niva AA 2 rufi AA 2 glab BB 2 punc CC 2 offi EE 2 aust FF 2 brac GG 2 gran BBCC 4 minu CCDD 4 alta HHKK 4 coar HHJJ 4 ridl Repeatmasker (>20 bp SSRs, <20% divergence) CAP3 assembly with SSR- BES add FPC information collapse to unique SSRs Yeisoo Yu Breeders Tools will be coming that will provide identified polymorphic SSR flanking primers with chromosome and pseudomolecule locations for the AA species. 27

28 Getting back to the Drill down in C-Map Display of the grain wt QTL Once the sequenced map has been drilled down sufficiently, load on the OMAP physical maps to locate the contigs and clones for that specific region that can be used as markers for breeding or positional cloning. In this example, use O. rufipogon FPC. Notice other species available. 28

mapping population Drill down on the sequenced map and into the physical

29 Alignment of the OMAP O. rufipogon FPC 2006 map Localization of the HGRWT QTL identified in the mapping population Drill down on the sequenced map and into the physical map. Contig 27 contains BAC clones mapping to the pseudomolecule and extending beyond to contain the QTL Ctg27 OR_CBa 054J23 005B01 29

SyMAP view of the two contigs that align to

mapping and Marker Assisted Selection and

30 SyMAP view of the two contigs that align to the end of the chromosome. 604 end sequences of the BACs can now be used to develop SSR markers for Fine mapping and Marker Assisted Selection and the BACs can be used for candidate gene identification and cloning. 30

31 AA genome Oryza species that currently have excellent FPC physical maps for development of Back cross populations: Oryza nivara Oryza rufipogon Oryza glaberrima AA Oryza species that are in process and will be available soon: Oryza barthii OMAP Cross Combinations at AGI AA x AA BB x BB CC x CC Reciprocal crossess of 9311(Indica) x Nipponbare (Japonica) Nipponbare x (O. glaberrima, O. glumaepatula, O. nivara, O. barthii, O. meridionalis, O. longistaminata, O. rufipogon) 9311 x (O. glaberrima, O. glumaepatula, O. nivara, O. barthii, O. meridionalis, O. longistaminata, O. rufipogon) O. punctata (IRGC ) x O. punctata ( Acc from Africa) O. officinalis (IRGC ) x O. officinalis (Acc from Asia) O. officinalis (IRGC ) x O. eichingeri (Acc from Africa) O. eichingeri (Acc from Africa) x O. eichingeri (Acc from Asia) BBCC x BBCC CCDD x CCDD O. minuta (IRGC ) x O. malaphuzaensis (Acc from India) O. minuta (IRGC ) x O. punctata (Acc from Africa) O. minuta (IRGC ) x O. minuta (Acc. from Asia) O. alta (IRGC105143) x O. latifolia (Acc from South America) O. alta (IRGC105143) x O. grandiglumis ( Acc from South America) GG x GG HHJJ x HHJJ O. granulata (IRGC ) x O. meyeriana (Acc from Asia) O. granulata (IRGC ) x O. neocaledonica (Acc from New Caledonia) O. ridleyi (IRGC ) x O. longiglumis (Acc from Africa) O. ridleyi (IRGC ) x O. ridleyi (Acc from Asia) HHKK x HHKK EE x EE P. coarctata (IRGC ) x O. schechteri (Acc from Indonesia) P. coarctata (IRGC ) x O. schechteri (Acc from Papua New Guinea) O. australiensis (IRGC ) x Annual and Perennial O. australiensis 31

COLLABORATION to use the OMAP data and resources for rice improvement We are interested in collaborating with breeders and geneticists for the development/utilization of diverse rice genes and for

32 COLLABORATION to use the OMAP data and resources for rice improvement We are interested in collaborating with breeders and geneticists for the development/utilization of diverse rice genes and for developing competitive rice cultivars that can exploit and use these wild Oryza genomes. Distribution of OMAP genomic resources from AGI Resource Center BAC clones and screening filters: species Library whole libraries individual clones screening filters O. brachyantha OB Ba O. nivara OR_BBa O. rufipogon OR_CBa O. punctata OP Ba Self O. supporting australiensis since 2003 OA_ABa O. alta OA_BBa O. glaberrima OG_BBa O. minuta OM Ba O. coarctata OC Ba O. officinalis OO Ba O. granulata OG_ABa O. ridleyi OR_ABa Total

Consideration for: Material Transfer Agreement IRRI's Policy on Intellectual Property Rights APHIS USDA Importation permit Phytosanitary certificate Requests for germplasm: Dr.

org OMAP voucher plants at IRRI Requests for OMAP Voucher plot materials: Dr. Darshan S.

33 Consideration for: Material Transfer Agreement IRRI's Policy on Intellectual Property Rights APHIS USDA Importation permit Phytosanitary certificate Requests for germplasm: Dr. Ruaraidh Sackville Hamilton Head, Genetic Resources Center International Rice Research Institute (IRRI) DAPO Box 7777 Metro Manila, Philippines OMAP voucher plants at IRRI Requests for OMAP Voucher plot materials: Dr. Darshan S. Brar Plant Breeding, Genetics & Biotechnology International Rice Research Institute (IRRI) DAPO Box 7777 Metro Manila, Philippines Future Directions - More AA genome OMAP s (O. barthii, O. longistaminata, O. meridionalis, O. glumaepatula) - Development of a rearrangement index for Oryza to identify the regions of difference - Molecular cytogenetic characterization of all OMAP genomes - Collaboration with breeders to develop Advanced BC populations with US cultivars x OMAP lines to the BC6F2 level for QTL & competitive cultivar development - Genome wide virtual SSR/SNP maps for all AA genome species - Establishment of I - OMAP o ABC/CSSL populations world wide o 6X draft sequencing of all OMAP accessions (~90 Gb) - Outreach to students and teachers in US rice regions 33

Glossary of terms BAC clone BAC Library Physical Map BAC Contig BAC end sequence A transformed E.

A collection of BAC clones that statistically represent an organisms genome.

A collection of BAC clones that are scientifically ordered and overlap in a tile.

directions. OMAP - Acknowledgements AGI/AGCoL Rod Wing HyeRan Kim Dave Kudrna Yeisoo Yu Jose L.

34 Glossary of terms BAC clone BAC Library Physical Map BAC Contig BAC end sequence A transformed E. coli bacterium that contains a large fragment of a genome ( kbp) that is cloned into a plasmid. A collection of BAC clones that statistically represent an organisms genome. The scientifically ordered collection of BAC clones such that every chromosome of an organism has an ordered set. A collection of BAC clones that are scientifically ordered and overlap in a tile. The DNA terminal sequence of a BAC clone initiated from the cloning vector and reading into the insert; both forward and reverse directions. OMAP - Acknowledgements AGI/AGCoL Rod Wing HyeRan Kim Dave Kudrna Yeisoo Yu Jose L. Goicoechea Meizhong Luo Raju Jetty Andrea Zuccolo Paul Sanchez Will Nelson Cari Soderlund OMAP Scott Jackson (Purdue) Lincoln Stein (CSHL) Bin Han (NCGR) Phillip San Miguel (Purdue) Doreen Ware (CSHL) Bonnie Hurwitz (CSHL) Darshan Brar (IRRI) Dave Mackill (IRRI) Nori Kurata (NIG) Susan McCouch (Cornell) David Galbraith (U Arizona) Olivier Panaud (U Perpignan) 34

The species of the genus Oryza and transfer of useful genes from wild species into cultivated rice, O. sativa

Breeding Science 60: 518 523 (2010) doi:10.1270/jsbbs.60.518 Review The species of the genus Oryza and transfer of useful genes from wild species into cultivated rice, O. sativa Kshirod K. Jena* 1) 1)