Selection of candidate genes (CGs) andWHEALBI molecular polymorphisms
for biomass production in [email protected]
Lodi: lab
BARPLUS kick-off meetingFiorenzuola d’Arda
12-13 May 2016
BARPLUS ideotype traits
PARTNERS AND PROPOSED TRAITS• Milano: photosynthesis, tillering and leaf angle• Potsdam: leaf size• Lleida: N uptake and partitioning CONFIDENTIAL
Candidate genes
Induced genetic VariationMutants
Natural geneticVariation
Allele mining from ExCap data
Phenotypic effects
FWD / REVWP1 WP2
CONFIDENTIAL
Presentation plan
1. The WHEALBI barley collection - Alessandro Tondelli
2. WHEALBI barley exome sequencing data - overview, state
of progress, data access - Laura Rossini
3. Allele mining on WHEALBI data - Agostino Fricano
4. BARPLUS candidate genes - all
CONFIDENTIAL
The WHEALBI barley collection
512 accessions:• Barley core subset from the IPK
• GABI-GENOBAR association panel
• IPK spring barley landrace association panel
• ERA-PG EXBARDIV association panel (history of European barley breeding)
• Parents of NAM and MAGIC populations
The WHEALBI barley collection
CONFIDENTIAL
6
• Genotype classification obtained from IPK (some data to be confirmed)
The WHEALBI barley collection
• 73 countries, Top 50%: Ethiopia,Turkey, Italy, Syria, Germany, Libya,Russia, Israel, Iran, France, India
• 213 have long/lat data
The WHEALBI barley collection
CONFIDENTIAL
Group by row type
2 rows(268 genotypes)
6 rows(244 genotypes)
The WHEALBI barley collection
CONFIDENTIAL
Laura Rossini
FP7 European Project
WP2 - Barley exome sequencing
CONFIDENTIAL
WP2: Gene sequence diversity revealedin wheat and barley genepool
Task 2.1 Task 2.2 Task 2.3
WHEALBI barley exome sequencing data
CONFIDENTIAL
WP2 - Barley work-plan
• Task 2.1 - Exome capture and sequencing of c. 500 barleyaccessions. Partners: PTP
• Task 2.2 - Barley QC sequences, deconvolute and establish robustpipelines for calling sequence variants. Partners: PTP
Expected output:List of robust molecular variants for each accession compared to the
exome capture design space template and described in the context ofthe emerging barley genome assembly.
Status of barley genome: a new assembly (pseudomolecules) has beenproduced by IPK - access still restricted but should be realeased thisyear. Waiting for new annotation.
WHEALBI barley exome sequencing data
CONFIDENTIAL
Library Preparation: Kapa protocol
Hybridization: pooled libraries hybridizedto the SeqCap EZ Oligo pool (47°C48h).
Bead Capture: Capture beads used to pulldown the complex of capture oligosand gDNA fragments.
Washing: Unbound fragments removed bywashing.
Amplification: Enriched fragment poolamplified by PCR.
Sequencing-Ready DNA: IlluminaHiSeq2000 100bp paired-ends runmodule
Targeted Region Capture Workflow-Nimblegen
WHEALBI barley exome sequencing data
CONFIDENTIAL
T2.1 - Exome capture and sequencing
Exome capture and resequencing of 512 barley accessions completed
Average 40 million reads per accessionAverage 28 million high quality reads per accession
WHEALBI barley exome sequencing data
CONFIDENTIAL
WHEALBI barley exome sequencing data
CONFIDENTIAL
Checks on ExCap data
• 67 samples with low yield due to failure of the exome capture protocol(experienced and reported also by IPK)
• 64 samples scheduled for re-running at PTP (DNAs received fromIPK April 2016).
• Remaining 445 samples cross-checked for heterozygosity andconcordance with IPK and JHI GBS and BeadExpress genotyping data
• Subset of 403 concordant samples with low heterozygosity werefurther processed for selection of a robust set of variants for GWASaccording to criteria provided by JHI
WHEALBI barley exome sequencing data
CONFIDENTIAL
T2.2 – Variant calling and convertingpseudochromosomes to full genome
GATK pipeline - parameters from IPK403 concordant samples64,523,315 total variants extracted - UNFILTERED VARIANTS
WHEALBI barley exome sequencing data
CONFIDENTIAL
WHEALBI barley exome sequencing data
CONFIDENTIAL
WHEALBI ExCap data availability
Data circulation is currently restricted to WHEALBI partners
A dedicated WHEALBI MTA will regulate access from otherconsortia such as BARPLUS. The interested parties willneed to specify the data they require access to, eg forBARPLUS sequence variants for specific gene models tobe listed.
WHEALBI barley exome sequencing data
CONFIDENTIAL
Acknowledgements
• Chiara Ferrandi - exome capture and sequencing• Francesco Strozzi, Ezequiel Nicolazzi - bioinformatics analyses
WHEALBI barley exome sequencing data
CONFIDENTIAL
Allele mining on WHEALBI data
A repository of SNP variants was created from WHEALBI ExCap data
Allele mining on WHEALBI data
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Wet lab and in silico validations of theWHEALBI SNP repository
* Singleton: allele found only in one individual
Allele mining on WHEALBI data
CONFIDENTIAL
The WHEALBI SNP repository includesmore than 2M SNPs
Key figures & facts•> 2M SNPs•SNPs in exons and flankingsequences (± 80 nt)•Number of singletons ~ 400K
Chr1H Chr2H Chr3H Chr4H Chr5H Chr6H Chr7H Total
SNPs 272,274 332,091 332,310 259,517 318,758 266,345 342,830 2,124,125
Allele mining on WHEALBI data
CONFIDENTIAL
The WHEALBI SNP repository enablesallele mining and haplotype discovery
Allele mining of ppd-H1
SNP in exons: 57Synonymous changes: 26Non Synonymous changes: 31
Haplotype network of ppd-H1 inferrredusing 31 non-synonymous SNP
mutations
Whe
albi
acc
essi
ons
Allele mining on WHEALBI data
CONFIDENTIAL
The WHEALBI SNP repository has beenenriched with the functional annotation of
variants
Allele mining on WHEALBI data
CONFIDENTIAL
Towards a more comprehensiveWHEALBI variant repository
Name Whealbi SNP repository v.1 Whealbi variant repository v.2
Variant type SNPs SNPs & InDels
Marker number >2M > 3M (expected)
Barley genomeannotation V.1 V.2
Automatedfunctional annotation
of variantsPartially unreliable More accurate
Imputation ofmissing data Available on request Only for SNPs
Status Available Q4 2016
Allele mining on WHEALBI data
CONFIDENTIAL
WHEALBI – CREA allele mining &diversity team
• Davide Guerra, Ph.D• Alessandro Tondelli, Ph.D• Agostino Fricano. Ph.D
Allele mining on WHEALBI data
CONFIDENTIAL
BARPLUS Candidate genes
PARTNERS AND PROPOSED TRAITS• Milano: photosynthesis, tillering and leaf angle• Potsdam: leaf size• Lleida: N uptake and partitioning
Leaf angle
•Erect leaves allow deeper penetration of light in the cropcanopy and have a higher leaf area index (single-side leafarea per unit of land area) which increases the capture oflight for photosynthesis and nitrogen use in dense plantings(Yang & Hwa Heredity 2008)•Small leaf angles in rice osdwarf4 and maize liguleless2mutants enabled higher-density planting increasingbiomass yield
BR and leaf angle inclination
A rice BR synthesis mutant has more erect leaves and increased yields
The more erect leavesmay cast less shadow onlower leaves, permittingmore photosynthesis
Reprinted by permission from Macmillan Publishers Ltd. Nature; Sakamoto, T., et al. (2006). Erect leaves caused bybrassinosteroid deficiency increase biomass production and grain yield in rice. Nat Biotech 24: 105-109, copyright 2006.
Sakamoto et al (2006) Nature Biotech 24:105
above-groundpanicle dry weight
Wild-typeOsdwarf4 mutant
Leaf angle candidate genes
•OsDWARF4 - NB two close paralogs in barley•Rice lc2, OsARF19•Maize liguleless1, liguleless2•Sorghum dwarf3 - NB pleiotropic effects•In progress
Leaf angle candidate genes
•OsDWARF4 - NB two close paralogs in barley•Rice lc2, OsARF19•Maize liguleless1, liguleless2•Sorghum dwarf3 - NB pleiotropic effects•In progress
CONFIDENTIAL
Tillering
•Tillers contribute to biomass production
Barley tilleringmutants
Hussien et al. 2014
*
*
* *
Hussien et al. 2014
The making of a tillerSAM
P5
AXM
Strigolactones inhibit shootbranching
Image courtesy RIKEN
Wild type SL-deficient
In mutant plantsunable to make SLs,many more shootbranches grow out
SL biosynthesispathway
•carlactone
•MAX3, D17, RMS5, DAD3:
•MAX4, D10 ,RMS1, DAD1:
•MAX1
•STRIGOLACTONES
•CCD7
•CCD8
•(P450)•MAX; Arabidopsis•D; rice•RMS; pea•DAD; petunia
•Thesereactions occur
in the plastid
•D27 (β-carotene-9-isomerase)
•Umehara, M., Hanada, A., Yoshida, S., Akiyama, K., Arite, T., Takeda-Kamiya, N., Magome, H., Kamiya, Y., Shirasu, K., Yoneyama, K., Kyozuka, J., andYamaguchi, S. (2008). Inhibition of shoot branching by new terpenoid plant hormones. Nature 455: 195-200.; Seto Y, Kameoka H, Yamaguchi S, Kyozuka J. (2012)Recent advances in strigolactone research: chemical and biological aspects. (in press). Alder, A., Jamil, M., Marzorati, M., Bruno, M., Vermathen, M., Bigler, P., Ghisla,S., Bouwmeester, H., Beyer, P., and Al-Babili, S. (2012). The path from β-carotene to carlactone, a strigolactone-like plant hormone. Science. 335: 1348-1351.
SAM
MOC1 LAX1
TAD1
MIP1 LAX2
AXM formaSonand maintenance
SL
Tiller budoutgrowth
Carotenoid
D17
D10
D27
MADS57 OsTB1
miR444a
GAmiR156
IPA1
NSP1 – NSP2BIOSYNTH
ESIS
SIGNALING
CK
PATTHIS1
??MAX1
D14
D3
D53
Hussien et al 2014 updated
BARPLUS Candidate genes
POINTS FOR DISCUSSION:• How to prioritize CGs for TILLING?
Possible criteria:– Demonstrated effects in increasing biomass when function lost– Single copy– Size/gene structure– Supporting evidence in barley (gene expression data,co-localization with known loci?)– Others?
• Once we have identified alleles and haplotypes from WHEALBI EXCAP data how can we link them to phenotypes?
