A barley root mutant collection for NGS-based fast-forward genetics Roberto Tuberosa Dept. of Agricultural Sciences University of Bologna, Italy 2 nd Plant Genomics Congress Kuala Lumpur, 19-20 March, 2015 mmmm
TILLMore: a barley mutant population in the cultivar Morex Cv. Morex NaN 3 based mutagenesis ca. 3,500 M 5 lines (originally 4,906 M 3 ) Database with phenotypes 19 genes tilled, 104 mutants identified (7 knockouts, 53 missenses) Mutation frequency: 1 mutation/480 kb (= 2.1 /Mb) www.distagenomics.unibo.it/tillmore Resource available for collaboration Talamè et al. 2008, Pl. Biotech. J. 6: 477-485
TILLMore and forward-genetics Huge amount of induced phenotypic variation is present in the TILLMore collection: 32.7% (1,605 of 4,906) of M 3 families showed a distinct phenotype, either fixed or segregating, in field-plot observations. Most frequent phenotypes: Plant/leaf color (12.2%), spike morphology (9.2%), plant height/vigor (7.0%). Likely underestimated! Efficient forward-genetics methods for mutant gene mapping and cloning were recently developed based on: High-density SNP-array profiling & Bulk Segregant Analysis (BSA) Mapping-by-sequencing approaches (ShoreMap, MutMap, or others) Applications in barley (Cloning many-noded dwarf, Mascher et al. 2014; HvPHYC, Pankin et al. 2014)
Fast forward-genetics approaches To reduce the noise from the foreground genome and prioritize mutations from WGS: 1. Outcrossing Large/small F2 pools Ggggg g SHOREmap (Schneeberger et al., 2009) NGM (Austin et al., 2011) 2. Backcrossing/selfing BC1/M3 pools MutMap (Abe et al., 2012) MutMap+ (Fekih et al., 2013) Ggggg g James et al. 2013
Why focusing on roots Thousands of loci affect root development and functions. In cereals, root architecture (including seminal roots) affects field performance through water and nutrient acquisition (Sanguineti et al. 2007; Chloupek et al. 2010, Singh et al. 2011, Uga et al. 2013; Canè et al. 2014; Cristopher et al. 2014; Lynch et al. 2014). No comprehensive collection of root mutants in cereals. Barley collection of root hairs mutants (c/o I. Szarejko). Natural variation has been investigated in collections of wild (H. spontaneum) (Grando, 1995) and cultivated barley (Naz et al. 2014).
Why focusing on roots Root architecture is now fully integrated in new crop ideotypes Steep, cheap and deep (Lynch 2013) Donald CM (1968) The breeding of crop ideotypes. Euphytica, 17: 385-403. Lynch J (2013). Steep, cheap and deep, an ideotype for maize roots. Annals of Botany, 112: 347-357.
Objectives To screen the barley TILLMore mutant collection for root mutants at seedling stage. To set up a forward-genetics protocol for fast mapping and cloning (root) mutants using TILLMore. To test the feasibility to use TILLMore as a functional genomic platform for NGS-based forward genetics in barley.
Materials and Methods
Screening TILLMore for root mutants Presently, 3,500 M 5 lines; 3,071 lines with enough seeds. 12 pre-germinated healthy seedlings from each TILLMore family. Paper-rolls: 2-L beakers, distilled water, growth chamber (16/8 h photoperiod, 24/22 C day and night, 8 days). Two additional paper-rolls for putative mutated families. Lines with robust phenotypes are being further screened in rhizotrons, grown at 45 + flat A3 scannerring. Bovina et al. (2011). Plant Genetic Resources, 9: 357-360.
Seminal roots (approx. five in Morex wt) Nodal (adventitious) roots
10 cm Testing for Mendelian inheritance Cross Analysed F 2 (no.) Observed Mutants/wild-types (no.) Expected mutants/wild-types (no.) c2 test 2588 x Proctor 304 66 238 43 261 0.30 (ns) 194 x Proctor 75 16 59 18.7 56.3 0.47 (ns) 5141 x Proctor 91 19 72 22.7 68.3 0.91 (ns) 5992 x Proctor 95 27 68 23.7 71.3 0.98 (ns) Sr-5992 Morex F2 of Sr5992 x Proctor
Proctor Morex Mutant SR5992 Mutant SR5992 BULK Short-root BULK Short-root BULK wt-root BULK wt-root Mapping observed mutants: SNP array-based BSA Mutant F 2 plants wt F 2 plants 1. iselect Infinium barley 9k array (Comadran et al. 2012 ): Analysis of genotype calls and Ɵ values (Ɵ = 2/π*arctan(Y/X) Proctor; Morex; Mutant line Bulk mutants (15 F 2 plants) Bulk wt (15 F 2 plants) 8 wild-type F 2 plants 8 mutant F 2 plants in double
Mapping observed mutants: NGS Additional exome-capture/ngs-based approaches are being tested in order to streamline the linking of a mutant with the underlying gene. 2. Exome Seq-based BSA (Gravitropic mutant 194) See Mascher et al. 2014 3. Exome Seq of the four mutant lines, once mutation has been narrowed down by genetics/bsa Wild type Morex Reference Mutant Mutant Cannot rely on Linkage Disequilibrium alone!
NGS Mapping-by-sequencing pipeline (in collaboration with IPK, U. Scholz group) Exome capture assay (Mascher et al. 2013). Sequencing: Illumina HiSeq2000 (2 x 100 bp reads). Alignment of reads to the WGS assembly of cv. Morex, IBGSC 2012 (BWA-MEM /Novosort). Variant calling by SAMtools mpileup/bcftools (filtering: call quality > 40, read depth > 10). Merging to POPSEQ map (Mascher et al. 2013) to define the genetic map position (and focus on the region detected by SNP array-based BSA).
Results
Images from second step (two additional paper-rolls for putative mutated families) 41 Morex 98 187 194 Morex Morex Morex 485 Morex 940 Morex 1851 Morex 3000 Morex
Coiled and root hairs 310 MOREX WT
Rhizotron screening for Geotropic 194 Geotropic 194 Morex
Results of root mutant screening 3,071 M 5 lines screened, 150 root mutants identified 64 lines (2.1%) were fixed (putatively homozygous) for root phenotype (3 reps). 86 lines showed a root phenotype and were still segregating. 5% of lines showed a root phenotype 18 89 6 17 3 6 6 2
Norm intensity (B) Results of the SNP array-based BSA By means of Genotype calls Δϴ value between bulk samples, +/+ vs -/- Norm intensity (A)
Chromosome 1H Chromosome 2H Chromosome 3H Chromosome 4H Chromosome 5H Chromosome 6H Chromosome 7H 2,496 SNPs were mapped between Morex and Proctor (iselect 9k barley map)
Short root F2 Short-root Wt-Root F2 F2 SCRI_RS_212956 (48.7 cm) SCRI_RS_195940 (67.3 cm) BULK SEGREGANT ANALYSIS for ShR1-5992 (Visualized using FlapJack) 19 cm Morex Proctor Bulk - - Bulk + +
38.8 cm 40.4 cm 54.4 cm 68.4 cm Short root 5992 SCRI_RS_200387 46.4 cm Mut Allele Freq. (P=0.01) Mut Allele Freq. (P=0.05) 1H 2H 3H 4H 5H 6H 7H 4 3.5 3 2.5 2 14 cm 29.6 cm 1.5 1 0.5 0 0 20 40 60 80 100 120 140 160
56.7 cm 57.2 cm 59.4 cm 71.7 cm Geotropic 194 SCRI_RS_134034 59.4 cm One marker tested on F2 plants. Association with phenotype was rejected! Mut Allele Freq. (P=0.01) Mut Allele Freq. (P=0.05) 1H 2H 3H 4H 5H 6H 7H 0.25 0.2 2.2 cm 0.15 0.1 15 cm 0.05 0 0 20 40 60 80 100 120 140
50.7 cm 54.8 cm 70.7 cm 76.6 cm Two markers tested on F2 plants. Association with phenotype was rejected! Short root 5141 SCRI_RS_129686 67.2 cm Mut Allele Freq. (P=0.01) Mut. Allele Freq. (P=0.05= 1H 2H 3H 4H 5H 6H 7H 0.3 0.25 0.2 0.15 0.1 15.9 cm 25.9 cm 0.05 0 0 20 40 60 80 100 120 140 160
Preliminary results: Mapping-by-sequencing Mutant 194 Mutant 5141 Mutant 2588 Mutant 5992 M194_Bulk+/+ M194_Bulk-/- N reads 94751042 109284804 95923558 92565184 71950544 84123414 Read mapped, I alignment, not duplicated 64626436 69755077 67313274 63716510 57815814 35001110 Cov. Of targeted capture regions (+/- 300 bp) 49552655 52565205 51691937 48999350 44326421 17105025 The variant call pattern (Mutant Allele Frequency) of short-root mutant 5141 targeted a gene-bearing contig assigned to the chr. 7H. G/A (expected transition from NaN 3 ) was concident with the peak of Δϴ between bulk samples (+/+ vs -/-). The targeted gene has been annotated as a root-length related gene in Arabidopsis. This gene was selected as primary candidate for further validation.
Summary 64 mutagenized M 5 lines (out of 3,071 lines. 2%) showed unambiguous seminal root phenotypes, were viable and fixed. Approx. 5% of line showed a seminal root phenotypes (similar to 6% rate observed by White et al. 2009, in cv. Optic). Most common phenotype was short roots (89 mutant lines) which is likely due to the large number of loci that govern root length. Several long roots, geotropic, coiled and root hairs mutants were also identified. BSA based on high-density SNP array enabled quick and inexpensive mutant mapping (down to 2.2-15.9 cm). Mapping can be completed with just two bulks (= two samples) of 15 plants each.
Summary How to go from mutants to genes in TILLMore? Just sequencing the mutation-carrying line! Full genome seq: yet undoable on a large scale. Exome-Seq: too many mutations per line (approx. 10,000 mutations, 100-200 coding and 10-20 knockouts per line) BSA and Exome Seq Targeted or Exome Seq of each mutant line once mutation has been narrowed down by genetics/bsa! Low mutation density in parent line should facilitate gene identification (expected: 1-3 knockouts per chromosome) Exome Seq-based BSA
Project 289300 EURoot Partner 9 UniBO Root angle in AM panel in durum wheat seedlings Seminal Root Angle ( ) Project 244374 DROPS Partner 12 UniBO DP045 DP034
From: Nazemi et al., 2015, submitted Variation for root aerenchyma in durum wheat Area of aerenchyma (AA) in nodal root cross sections of 10 durum wheat cultivars
DW Contrasting NILs for root QTLs in maize root-yield-1.06 root-aba1-2.04 120 (--) 129 (++) Os (--) Os (++) 250.0 200.0 BB 197.3 150.0 100.0 50.0 80.3 47.4 102.4 0.0 120 (--) 129 (++) OS-- OS++ For yield, see Landi et al. (2010, J. Exp. Bot.) For yield, see Landi et al. (2007, J. Exp. Bot.)
Many thanks to: DipSA - University of Bologna Plant Genetics Silvio Salvi Riccardo Bovina Sara Giulia Milner Valentina Talamé Carlos Busanello Simona Corneti Sandra Stefanelli Italian Ministry of Research EU FP7 EUROOT IPK - Gatersleben Nils Stein, Axel Himmelbach Uwe Scholz, Martin Mascher