Oat Genomics at IBERS
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1 Oat Genomics at IBERS
2 Minor crop major genome 17Gb 2n=6x 5.1Gb 2n=2x 13Gb 2n=6x 2n=42 3.3Gb 0.43Gb
3 Major genome minor budget >$100M <$1M
4 Why genomics? Aka Why does this take so long and cost so much? REPEATS GENES REPEATS A. Easy things B. Avena AvKB9 EINMITEe WHAM-like intrt AvKB9 SABRINA-like intrt ANSA1 R173-like RTN ANSA1-like Hairpins EINMITEc MITEfuxi CACTA? SHERLOCK EINMITEg minicacta EINMITEh CACTA-TN EINMITEi intrt? Unknown ANSA1 LTR fuxi-full? MITEfuxi As14 tos17 HELITRON? degenerate CACTA COPIA Fourf DQ bp Sad1 GENES Sad2
5 Sad1 SUB-GENOME Y Sad2 Sad1 SUB-GENOME X Sad2 Sad1 SUB-GENOME Z Sad2
6 Non-genic too variable for a reference? (2 metaphase spreads) Comparison of maize bronze region
7 GbS-001 Sad1 SUB-GENOME Y Sad2 Random samples of polymorphisms will often target non-genic areas (repeat derived?) Sad1 SUB-GENOME X Sad2 - Need a reference map of genes to align to marker maps Sad1 GbS-002 SUB-GENOME Z Sad2
8 Synteny conserved gene content
9 Synteny conserved gene content* *may contain artefacts
10 Genome zippers
11 Genome zippers a 4b 4c
12 Avena zipper - Genetic Mapping Diploid species A. strigosa A. atlantica x F1 dense genetic map 2772 markers selfing 186 F6 individuals (RILs) SSRs and 3000 'DArT-seq' codominant markers, 92 RILs
13 Avena zipper - NGS based mapping anchor contigs based on parent-of-origin in RILs
14 NGS based mapping (POPSEQ) Figure A. Methods used to place sequences on the genetic map. (A) Placement of scaffolds. Out of a total of scaffolds (16.2%) are placed by at least one method. (B) Placement of high confidence gene models. Of genes, (94.1%) are placed by at least one method.
15 Transcriptome database, 11 tissues assembled and annotated
16 Synteny and lineage-specific evolution of key genes Synteny plot of reciprocal best blastp hits, showing base pair position in (A) brachypodium, (B) rice, (C) sorghum and (D) barley versus genetic map position in A. atlantica with positions of candidate CslF genes and their likely ortholog(s) shown with black circles. Orthology was based on gene phylogeny.
17 Additional reference genomes insularis DC (>20x cover) Lagurus outgroup longiglumis A damascena pre-a/d? canariensis pre-a/d? ventricosa - C Provides full Avena gene set, comparative resource for functional analysis, proxies for hexaploid sub-genomes
18 Hexaploid sequences can be seen to be derived from divergent sub-genomes software cannot always resolve into correct haplotypes - Diploid progenitors help software - Ideally physical sorting of templates?
19
20 Histogram of fluorescence intensity (flow karyotype) obtained after flow-cytometric analysis of 4,6-diamino-phenylindole stained chromosomes of Morex. Klaus F.X. Mayer et al. Plant Physiol. 2009;151: by American Society of Plant Biologists
21 FISHIS - Fluorescent In Situ Hybridisation in Suspension A R4 (ACT)5-FITC labelled FISHIS sorted chromosomes ( R4) (ACT)5-fitc FISH on metaphase spread B FISHIS - (ACT)5 labelled suspension C GBS tag content of FISHIS fraction R4 R4 Unlabelled chromosome suspension Debora Giorgi and Sergio Lucretti, ENEA, Rome
22 Expected synteny of R8, R9 and R3 fractions
23
24 Guide N50 Max Total Scaffold None 331bp 7.5kb 640k 5k N C-genome 465bp 47.1kb 593k 688k N A-genome 2.23kb 49.4kb 722k 72902k N Assemblies helped significantly by using diploid sequence as guide
25 Novel population development; NAM (Nested Association, common single parent) Spring Winter Assiniboia Husky Makita Canyon Pl F x FIRTH PI CIav 5332 PI Dancer PI Ogle Sol-Fi PI Freja SA99572 Hi-Fi PI Pol PI PI PI PI TAM O-301 Matilda Maldwyn Emotion PI PI PI Buffalo Harpoon Tardis 96-21Cn19 Millennium Mascani Brochan Bulwark Greywinter S172 S147 Hendon Lexicon Solva Racoon Krypton Expression Pendragon Grafton Fusion Penderi Balado Kingfisher Gerald 00-61Cn3 PI Coefficient Ethiopian tetraploid Common parent, Firth, European spring F7s, F8s of 600 lines grown in field GbS mapping underway POPSEQ?
26 GENE CONTENT & EXPRESSION GENOME ASSEMBLIES & CONTIG MAPPING COMPARATIVE RESOURCES tissues tissues tissues 11 tissues tissues tissues tissues 11 tissues tissues tissues tissues tissues tissues 11 tissues tissues NAM tissues tissues 11 tissues 11 Grain tissues & meristem ATL X STRIG ZIPPER FIRTH FISHIS FIRTH FISHIS FIRTH FISHIS FIRTH X damascena canariensis longiglumis VENTRICOSA CONTIGS AVENEAE SPP GBS dbs
27 GENE CONTENT & EXPRESSION GENOME ASSEMBLIES & CONTIG MAPPING COMPARATIVE RESOURCES 11 tissues 11 tissues 11 tissues 11 GRAIN tissues 11 tissues 11 tissues 11 tissues 11 tissues 11 tissues ATL X STRIG ZIPPER damascena canariensis longiglumis VENTRICOSA CONTIGS 11 tissues 11 tissues 11 tissues 11 tissues 11 tissues 11 tissues 11 NAM tissues 11 tissues 11 Grain tissues & meristem FIRTH FISHIS FIRTH FISHIS FIRTH FISHIS FIRTH X AVENEAE SPP GBS dbs Phenotype
28 GENE CONTENT & EXPRESSION GENOME ASSEMBLIES & CONTIG MAPPING COMPARATIVE RESOURCES STEM 11 tissues 11 tissues 11 tissues 11 tissues 11 tissues 11 tissues 11 tissues 11 tissues 11 tissues ATL X STRIG ZIPPER damascena canariensis longiglumis VENTRICOSA CONTIGS 11 tissues 11 tissues 11 tissues 11 tissues 11 tissues 11 tissues 11 NAM tissues 11 tissues 11 Grain tissues & meristem VALIDATE, REFINE TRAIT SELECTION & BREEDING STRATEGY FIRTH FISHIS FIRTH FISHIS FIRTH FISHIS FIRTH X AVENEAE SPP GBS dbs
29 APPLIED ARTIFICIAL Genetic resources for functional genomics 2500 azide treated lines (w/jic, 2013) TILLING - identify extreme alleles to confirm link with phenotype (diploid) 600 F7 from 15 populations (2014) NAM - compare alleles in related or near-identical backgrounds 600 S5 from 8 intercrossed parents (2015) as required MAGIC - compare allele in multiple related backgrounds Breeding panels - identify novel alleles in agronomically adapted backgrounds as required (>150 diploids, >1500 landraces etc) Diversity panels - identify novel alleles in environmentally adapted backgrounds
30 DNA stocks from viable and dead germplasm IBERS landrace panels
31 Identify likely progenitor populations
32 Identify adaptations missing from progenitors
33 Environmental adaptation or population structure?
34 IBERS resources funded by public/private collaboration, linked with other public funded researchers and projects - Aim to integrate with T3, metabolite dbs, et al
35 ENEA IBERS Rob Vickerstaff - Bioinformatics Catherine Howarth - Mapping, model populations, trait dissection Maciej Bisaga RNAseq, current LINK project Sandra Prusaka, Chris Creavey transcriptome database Adriana Ravagnani Pathway annotation Matt Hegarty - Genomics facility Athole Marshall - Head of Public Good Plant Breeding Debora Giorgi, Anna Farina, Sergio Lucretti FISHIS CRA Primetta Faccioli mirna analysis AAFC Ottawa Nick Tinker GbS Laval Brian Boyle GbS UNCC Schlueter lab hexaploid references
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