2013 Cornell's Plant Breeding and Genetic Seminar Series
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2013 Cornell's Plant Breeding and Genetic Seminar Series
- 1. Current Topics on Cassava Genetics Research and Future “Omics” Perspective Becerra Lopez-Lavalle L.A., Duitama J., Perera C., Ovalle T., Castillo K., and J. Tohme CIAT’s Annual Program Report – Cali 14 August 2013
- 2. Current Research Enhancing Cassava productivity through more targeted use of global genetic diversity Next-generation sequencing Functional Genetic for high quality cassava: pVA and Starch synthase pathways Whitefly control: a durable solution to cassava-disease pandemics (Africa and Asia) Whitefly resistance Carotenoid Synthase Pathway A 25 µg Total Carotenoid Content B β-OHase Zeaxanthin LCYB LCYB + LCYE ZDS + CRTISO PDS + Z-ISO β-carotene Lycopene CMC-40 ζ-carotene Phytoene GGPP C Lutein PSY 15µg D β+ε-OHase α-carotene MEcu 72 Chromoplast Whitefly molecular identification 99 75% Amylopectin A Bemisia tabaci biotypeA KF059958 CIAT 99 30 New World Bemisia tabaci AY521259 Aleurolobus marlatti JQ340188 Asia Siphoninus philyreae JQ340199 Aleurocanthus inceratus HM150621 Aleurocanthus spiniferus JQ340176 45 Asia Crenidorsum micheliae JQ340190 97 C Aleurotrachelus trachoides KF059957 CIAT 98 Aleurothrachelus socialis KF059953 CIAT 89 88 New World D Aleurothrixus floccosus KF059956 CIAT Aleuroclava sp JQ340187 ADP 93 Asia Dialeurodes hongkongensis JQ340195 E New World Trialeurodes variabilis KF059962 CIAT Trialeurodes vaporiariorum KF192508 CIAT 99 G-IV Dialeurodes citri JQ340193 98 G-III Dialeuropora JQ340197 73 New World G-V 68 ADPG B G-II 99 Aleyrodinae ATP PPi F 100 Trialeurodes vaporariorum AY521265 GBSSI Aleuronudus melzeri KF059955 CIAT Aleurodicinae G1P Asia / Asia-Minor Bemisia tabaci biotypeB KF059959 CIAT 96 DE SBEII SBEI SSIII SSII SSI Bemisia tabaci JN410741 Bemisia tabaci JN410738 81 100 G-I Starch Synthase Pathway 25% Amylose G Aleurodicus dugessi AY521251 46 99 Lecanoideus floccissimus KF059960 CIAT H Aleurodicus dispersus KF059954 CIAT 95 99 Aleurodicus dispersus EU581838 I 75 Aleurodicus dispersus EU581837 Amyloplast Drosophila melanogaster ADW61439 100 Drosophila melanogaster ADW61333 Drosophila melanogaster AGE13862 0.20 0.15 0.10 0.05 0.00 G-VI Metabolomic profiling Asia
- 3. Our goal • Undertake world-ranking plant biology and cassava improvement research … • To support small holder farmers, by expanding and diversifying the use of cassava as renewable sources of … • High-value food, feed and industrial biomaterials (carbohydrate and fiber)
- 4. Global Cassava Diversity Enhancing Cassava productivity through more targeted use of global genetic diversity 5000 to 7000 AC (Piperno and Holst 1998; Rouse 1953 Next-generation sequencing M. aesculifolia Rogers 1965; Rogers and Fleming 1973; Rogers and Appan 1973) Metabolomic profiling 1800 AC to 1532 BC Ugent et al. 1986 1 ,2 5 0 AC NO DATED Allem et al. 1994 Allem et al. 2001 and Allem 2002
- 5. Global Cassava Diversity Enhancing Cassava productivity through more targeted use of global genetic diversity Next-generation sequencing Metabolomic profiling Asia Africa
- 6. Global Cassava Diversity Enhancing Cassava productivity through more targeted use of global genetic diversity Next-generation sequencing Metabolomic profiling GWAS 1’000,000 bp = 50 to 100 genes http://www.vasculairegeneeskunde.nl/ivg-2010-presentatie-4
- 7. Global Cassava Diversity: Genomics Enhancing Cassava productivity through more targeted use of global genetic diversity Strategy Next-generation sequencing Sampling • Selection of relevant breeding genotypes Metabolomic profiling • Geographic distribution RAD-SEQ Bioinformatic Pipeline Pop. GENOMICS Whole Genome Region/Window • Mapping to REF-GENOME • Pop. Structure • Adequate data filtering • Pop. diversity • Variant detection and genotyping (NGSEP) • Differential and random matting estimates • Nucleotide diversity • Tajima’s D • Kernell smoothed FSTs SNP-KIT Genotype validation/ Identification • Identification of highly informative SNPs to discriminate among cassava accessions
- 8. Global Cassava Diversity: Historical Data Enhancing Cassava productivity through more targeted use of global genetic diversity Next-generation sequencing • Metabolomic profiling 1,900 Experimental nurseries • Nearly 15’000,000 data points
- 9. Global Cassava Diversity: CIAT’s breeding Enhancing Cassava productivity through more targeted use of global genetic diversity • • Champion genotypes: MCol22, MCol1505, MCol1684, MBra12. • Next-generation sequencing Identification of the 139 founders CIAT’s cassava breeding program Identification of 25 trait-specific genotypes to construct the Cassava-Basic Metabolome Profile Founder freq. Metabolomic profiling X Genotypes
- 10. Global Cassava Diversity: Genomics Enhancing Cassava productivity through more targeted use of global genetic diversity Sampling Next-generation sequencing CASSAVA (N=139) Metabolomic profiling MEX (7) CUB (6) PTR(5) RDOM (2) GUA (13) CR (10) PAN (5) COL (29) VEN (9) ECU (6) BRA (5) PER (9) BOL (3) PAR (2) ARG (7) CH (2) TAI (3) MAL (4) IND (2) AFR(4) BREED (4) WILD (N=24) FLAV (3) - M. flabellifolia ALT- M. alutacea BLO- M. brachyloba CTH- M. carthaginensis GUT- M. guaranitica PIL- M. pilosa S1WPD - M. walkereae TST - M. tristis PNT - M. Pentaphylla ORB - M. orbicularis PEL - M. peltata
- 11. Global Cassava Diversity: Genomics Enhancing Cassava productivity through more targeted use of global genetic diversity Next-generation sequencing Metabolomic profiling Bioinformatic’s Pipeline
- 12. Global Cassava Diversity: Genomics Bioinformatics' Pipeline • Align reads to reference • Bowtie2-2.1.0-beta7 • Sort alignments • Piccard’s Tools • Calling SNPs • NGSEP – CIAT’s • Calling Genotypes • Allele and genotypes counts / Frequencies • Java customized programs • Statistics & Annotations using GFF from Phytozome • Comparing genotypes across populations. • Statistics & Annotations using GFF from Phytozome • Structure • Power Marker • TASSEL • CIAT costumed programs
- 13. Global Cassava Diversity: Genomics Enhancing Cassava productivity through more targeted use of global genetic diversity Next-generation sequencing Population Structure • STRUCTURE v.2.3.4 (Pritchard, Stephens, & Donnelly, 2000). Admixture model with correlated of allele frequencies. Number of populations (K) from 1 to 10. Metabolomic profiling Burn-in period on 10000 and MCMC Reps 20000. • ΔK method (Evanno 2005) implemented in the software STRUCTURE HARVESTER (Earl et al., 2012) to ascertain the most likely value of K. • Membership coefficients (Q scores) >= 0.9 used to assign individuals to clusters. • With and without sampling locations as a prior (Country of origin)
- 14. Global Cassava Diversity: Genomics Enhancing Cassava productivity through more targeted use of global genetic diversity Next-generation sequencing Population Genomics • PowerMarker v.3.25 (Liu and Muse 2005) • Unbiased estimate of gene diversity (He) • (Nei’s 1983) F-statistics to quantify genetic differentiation and random mating within and between hierarchical levels (Wright 1983) FST (coancestry coefficient or fixation index) proportion of the total genetic variance contained in a subpopulation. Metabolomic profiling FST = 1 - (HS / HT) FIS (Inbreeding coeffficient) proportion of the genetic variance of the subpopulation contained in a individual FIS = 1 - (HI / HS) • Frequency matrix and genetic distance • followed by cluster analysis with the weighted neighbor-joining (NJ) algorithm. Relationships among groups were visualized in a dendogram edited in MEGA Vs.5.1, and colored by structure clusters.
- 15. Global Cassava Diversity: Genomics Sliding Windows approach • Genes critical to cassava domestication can be identified from their wild relatives. Genomic regions can be identified as outliers in a genome scan increasing the power to detect selection in natural populations • Genomic regions with increased or decreased differentiation across populations Continuous distribution across the genome • diversifying or stabilizing natural selection • identify candidate genes, pathways and alleles for targeted functional analysis • Nucleotide Diversity (π) • Heterozygosity • Kernell – Smoothed FST • Tajima’s D • ROD= Reduction of diversity Level of Polymorphism TEST of departures from Neutral Equilibrium: Chromosomal position
- 16. Global Cassava Diversity: Genomics Sliding Windows approach Genomic regions can be identified as outliers in a genome scan increasing the power to detect selection in natural populations Limited software to perform genome windows analysis across the genome TASSEL • Only accept data organized by • STACKS ??? • chromosomes Don’t allow for populations comparison Do not calculate divergence estimates: FST
- 17. Global Cassava Diversity: Genomics DEVELOPING A CORE SET OF SNPs FOR VALIDATION AND IDENTIFICATION OF CASSAVA ACCESSIONS High Quality SNPs Technical criteria for SNPtype™ Assays in Fluidigm. Highly Informative SNPs shared between WGS and RAD-seq No SNPs around 40bp SNP/Scaffold Filters: Repetitive regions and CNVs GC content from 40 to 65 Mínimum MAF 0.2/0.15 Quality Score: Higher than 40 More than 10 homozygous differences
- 18. Global Cassava Diversity: Genomics VARIATION IN THE RESTRICTION SITES UNDERESTIMATE HETEROZYGOSITY IN RAD SEQUENCING IGB vs.7.03
- 19. Global Cassava Diversity: Genomics Genetic distance and Structure between Cassava and wild relatives Segregating SNPs 34 M. tristis 1342 M. guaranitica 1700 M. walkereae • • • • • Higher diversity in Wild (0.33) than in Cassava (0.12) Most wild Cassava relatives highly differentiated for cultivated Cassava (Fst=0,65 W-C and 0,96 K between clusters). More similar wild relatives: FLA, ALT, TST More distant wild relatives: S1WPD1 Fis higher in Wild Manihot species (0.98).
- 20. Enhancing Cassava productivity through more targeted use of global genetic diversity Cuba - Panama Colombia – Venezuela Guatemala Next-generation sequencing Guatemala – Costa Rica 200 genotypes Colombia – Ecuador - Peru Global Cassava Diversity: 3rd Output Metabolomic profiling • 700 LAC + 300 Africa towards the global goal. • Designed 180 SNPs for variety identification and pedigree reconstruction (underway).
- 21. Global Cassava Diversity: Metabolomics Enhancing Cassava productivity through more targeted use of global genetic diversity Next-generation sequencing Metabolomic profiling Materials for Metabolomic profiling Genotype 1 BRA488 2 DOM2 3 COL2017 4 PAR36 5 MPER183 6 MCOL2436 7 MECU72 8 MCUB74 9 CUB23 10 PAN139 11 TMS604444 12 TME3 13 TMS305555 14 BRA1A 15 MCOL1505 16 MCOL1522 17 GUA35 18 CUB25 19 MVEN25 20 MCOL22 21 MGUA85 22 MVEN77 23 MCOL113 24 MCOL638 25 MCOL1684 Trait Clones High Cianide Content 6 High Dry Matter Content 6 High Sugar Content 6 High Amilose Content 6 High PPD Tolerance/Frog Skin disease/Low Carotene 6 High Carotene/Trips Susceptible 6 WF Resistance/Bacteriosis Susceptible 6 High Culinary Quality/Z06 adaptation 6 Waxi Potential 6 Trips Resistance 6 Transformacion 6 CMD 6 CMD 6 High carotene 6 Z01 nd Z04 adaptation 6 Z05 adaptation 6 Low Sugar Content 6 Low Amilose Content 6 Low Culinary Quality 6 PPD susceptible 6 Adaptation Z02 6 Drought tolerance 6 Adaptation Z06 6 Bacteriosis resistance 6 Z03 adaptation 6 150
- 22. pVA and Starch: Why Functional Genetic for high quality cassava: pVA and Starch synthase pathways Amyloplast vs. Chromoplast Carotenoid Synthase Pathway 75% Amylopectin 25 µg Total Carotenoid Content SBEIIDE SBEI SSIII SSII SSI β-OHase Zeaxanthin LCYB LCYB + LCYE ZDS + CRTISO PDS + Z-ISO ATP PPi ADPG G1P 75% Amylopectin 25 µg Total Carotenoid Content β-carotene β-OHase Lycopene Zeaxanthin 15µg Zeaxanthin LCYB LCYB + LCYE ZDS + CRTISO PDS + Z-ISO ADPG ADP 25 µg Total Carotenoid Content Amyloplast ATP PPi ADPG G1P ADP ADP ZDS + CRTISO PDS + Z-ISO Amyloplast 25% Amylose G1P G1P ADPG Amyloplast 15µg β-OHase Zeaxanthin β-carotene Lycopene ζ-carotene Phytoene GGPP Chromoplast Lutein PSY 15µg 25 µg Total Carotenoid Content β+ε-OHase α-carotene β-OHase Zeaxanthin Chromoplast LCYB LCYB + LCYE ZDS + CRTISO PDS + Z-ISO ADP 25 µg Total Carotenoid Content β-carotene β-OHase Lycopene Lutein ZDS + CRTISO PDS + Z-ISO 15µg β+ε-OHase 25% Amylose Chromoplast Zeaxanthin LCYB LCYB + LCYE ζ-carotene Phytoene GGPP GBSSI GBSSI 25% Amylose LCYB LCYB + LCYE ZDS + CRTISO PDS + Z-ISO Chromoplast β+ε-OHase α-carotene PSY β-carotene Lycopene ζ-carotene Phytoene GGPP α-carotene Lutein PSY 15µg β+ε-OHase β-carotene ADPG G1P ADPG G1P ADP ZDS + CRTISO PDS + Z-ISO G1P ZDS + CRTISO PDS + Z-ISO ζ-carotene Phytoene GGPP PSY G1P ADP 25% Amylose Amyloplast Amyloplast β+ε-OHase α-carotene Chromoplast Starch Synthase Pathway 75% Amylopectin DE SBEII SBEI SSIII SSII SSI ATP PPi ADPG ADP GBSSI 25% Amylose Amyloplast β-carotene ZDS + CRTISO PDS + Z-ISO Lycopene PSY Lutein PSY β+ε-OHase α-carotene β+ε-OHase α-carotene Chromoplast Zeaxanthin LCYB LCYB + LCYE ZDS + CRTISO PDS + Z-ISO 25 µg Total Carotenoid Content β-carotene β-OHase Lycopene Zeaxanthin LCYB LCYB + LCYE ζ-carotene Phytoene GGPP Lutein PSY Lutein ZDS + CRTISO PDS + Z-ISO 15µg β+ε-OHase β+ε-OHase α-carotene PSY Lutein 15µg β+ε-OHase α-carotene Chromoplast Chromoplast Chromoplast Chromoplast β-OHase Zeaxanthin LCYB LCYB + LCYE ZDS + CRTISO PDS + Z-ISO Lutein β-carotene Lycopene ζ-carotene Phytoene GGPP α-carotene 25 µg Total Carotenoid Content Zeaxanthin β-carotene Lycopene ζ-carotene 15µg 25 µg Total Carotenoid Content β-carotene PSY 25 µg Total Carotenoid Content β-OHase Lycopene β-OHase Zeaxanthin LCYB LCYB + LCYE ζ-carotene Phytoene GGPP Lutein ZDS + CRTISO PDS + Z-ISO 15µg β+ε-OHase β+ε-OHase α-carotene α-carotene PSY Zeaxanthin LCYB LCYB + LCYE β-carotene ZDS + CRTISO PDS + Z-ISO Lycopene ζ-carotene Phytoene GGPP Lutein 15µg PSY Lutein 15µg β+ε-OHase α-carotene Chromoplast Chromoplast β-carotene Lycopene ζ-carotene Phytoene GGPP β+ε-OHase α-carotene Chromoplast Lutein 15µg 15µg β-OHase Zeaxanthin β-carotene Lycopene ζ-carotene 15µg Chromoplast β-carotene Lycopene ζ-carotene Phytoene GGPP ζ-carotene Phytoene GGPP 25 µg Total Carotenoid Content β-OHase PSY GBSSI 25% Amylose Lutein Zeaxanthin LCYB LCYB + LCYE Zeaxanthin β+ε-OHase α-carotene Chromoplast β-OHase PSY ADP Amyloplast β-OHase β-OHase LCYB LCYB + LCYE ZDS + CRTISO PDS + Z-ISO 15µg Chromoplast 25 µg Total Carotenoid Content LCYB LCYB + LCYE ZDS + CRTISO PDS + Z-ISO Phytoene GGPP β+ε-OHase α-carotene 25 µg Total Carotenoid Content LCYB LCYB + LCYE ZDS + CRTISO PDS + Z-ISO Phytoene GGPP ADPG Amyloplast 25 µg Total Carotenoid Content β-carotene Lycopene ζ-carotene Lutein PSY β+ε-OHase α-carotene Lutein 15µg Amyloplast Amyloplast 75% Amylopectin SBEIIDE SBEI SSIII SSII SSI ATP PPi G1P 25% Amylose ZDS + CRTISO PDS + Z-ISO Phytoene GGPP Lutein 15µg Zeaxanthin β-carotene Lycopene ζ-carotene Phytoene PSY GBSSI 25% Amylose 75% Amylopectin SBEIIDE SBEI SSIII SSII SSI PSY β-OHase ADP 25% Amylose 25% Amylose Amyloplast ADPG LCYB LCYB + LCYE β-carotene Lycopene ζ-carotene Phytoene GGPP β+ε-OHase α-carotene 25 µg Total Carotenoid Content LCYB LCYB + LCYE ZDS + CRTISO PDS + Z-ISO ADPG GGPP GBSSI ATP PPi ZDS + CRTISO PDS + Z-ISO Lutein 15µg Chromoplast Amyloplast 75% Amylopectin SBEIIDE SBEI SSIII SSII SSI ATP PPi ADPG ADP GBSSI GBSSI PSY GBSSI 25 µg Total Carotenoid Content Zeaxanthin Chromoplast ADPG GBSSI G1P LCYB LCYB + LCYE Lycopene ζ-carotene Phytoene GGPP ADP β-OHase Zeaxanthin 25% Amylose G1P ADP ZDS + CRTISO PDS + Z-ISO ADPG β+ε-OHase α-carotene ATP PPi G1P ATP PPi ADPG GBSSI 25% Amylose 25 µg Total Carotenoid Content β-OHase β-carotene Chromoplast 75% Amylopectin SBEIIDE SBEI SSIII SSII SSI SBEIIDE SBEI SSIII SSII SSI ATP PPi G1P ADP 15µg G1P 15µg Chromoplast Amyloplast 75% Amylopectin SBEIIDE SBEI SSIII SSII SSI ADPG Zeaxanthin ATP PPi Lutein PSY β+ε-OHase α-carotene GBSSI 25% Amylose Amyloplast 75% Amylopectin SBEIIDE SBEI SSIII SSII SSI ATP PPi G1P 25 µg Total Carotenoid Content β-OHase LCYB LCYB + LCYE ADP GBSSI 25% Amylose Amyloplast ζ-carotene ζ-carotene Phytoene GGPP Lutein 15µg ADPG SBEIIDE SBEI SSIII SSII SSI β-carotene Lycopene Chromoplast 25 µg Total Carotenoid Content Zeaxanthin LCYB LCYB + LCYE β-carotene Lycopene ATP PPi ADP GBSSI 25% Amylose 75% Amylopectin β-OHase Zeaxanthin LCYB LCYB + LCYE 75% Amylopectin SBEIIDE SBEI SSIII SSII SSI ATP PPi ADPG GBSSI 25% Amylose 25 µg Total Carotenoid Content β-OHase 75% Amylopectin SBEIIDE SBEI SSIII SSII SSI ATP PPi ADP α-carotene Chromoplast 25 µg Total Carotenoid Content 75% Amylopectin SBEIIDE SBEI SSIII SSII SSI ATP PPi G1P Lycopene Amyloplast Amyloplast 75% Amylopectin SBEIIDE SBEI SSIII SSII SSI ADP G1P Chromoplast 15µg ADPG ADP Amyloplast Amyloplast Phytoene PSY G1P ADPG GBSSI 25% Amylose 25 µg Total Carotenoid Content Lutein PSY β+ε-OHase ATP PPi ATP PPi G1P ADP GBSSI 25% Amylose β-carotene Lycopene ζ-carotene Phytoene GGPP β+ε-OHase α-carotene Lutein PSY Chromoplast Zeaxanthin 15µg α-carotene 75% Amylopectin SBEIIDE SBEI SSIII SSII SSI SBEIIDE SBEI SSIII SSII SSI ADPG ADP Lutein β+ε-OHase β-OHase LCYB LCYB + LCYE ZDS + CRTISO PDS + Z-ISO Lutein PSY β+ε-OHase α-carotene Phytoene GGPP Chromoplast 75% Amylopectin β-carotene Lycopene ζ-carotene Phytoene GGPP 15µg Lycopene ζ-carotene GBSSI 25% Amylose ATP PPi ATP PPi Zeaxanthin β-carotene α-carotene LCYB LCYB + LCYE ZDS + CRTISO PDS + Z-ISO Lutein PSY Zeaxanthin β-carotene 25 µg Total Carotenoid Content Zeaxanthin β-carotene Lycopene ζ-carotene Phytoene GGPP β-OHase LCYB LCYB + LCYE ZDS + CRTISO PDS + Z-ISO ADP SBEIIDE SBEI SSIII SSII SSI Amyloplast GGPP ZDS + CRTISO PDS + Z-ISO 25 µg Total Carotenoid Content Lutein β+ε-OHase α-carotene 75% Amylopectin β-OHase Lycopene ζ-carotene 15µg β-OHase Zeaxanthin LCYB LCYB + LCYE Amyloplast β-carotene Lycopene ζ-carotene 15µg Chromoplast ADPG Amyloplast 75% Amylopectin SBEIIDE SBEI SSIII SSII SSI 25 µg Total Carotenoid Content LCYB LCYB + LCYE ZDS + CRTISO PDS + Z-ISO Phytoene β-OHase Zeaxanthin PSY PSY 25 µg Total Carotenoid Content β-OHase LCYB LCYB + LCYE ZDS + CRTISO PDS + Z-ISO Phytoene GGPP β+ε-OHase α-carotene GGPP Chromoplast 25 µg Total Carotenoid Content 25% Amylose ATP PPi 75% Amylopectin ZDS + CRTISO PDS + Z-ISO β-carotene Lycopene ζ-carotene 15µg Chromoplast Lutein β+ε-OHase α-carotene Chromoplast 25 µg Total Carotenoid Content Lutein PSY α-carotene SBEIIDE SBEI SSIII SSII SSI GBSSI G1P ZDS + CRTISO PDS + Z-ISO Phytoene GGPP 15µg β+ε-OHase ADP Amyloplast Zeaxanthin LCYB LCYB + LCYE Lycopene ζ-carotene Lutein PSY 75% Amylopectin ADPG G1P GBSSI 25% Amylose Amyloplast Zeaxanthin β-carotene 15µg β+ε-OHase ADP β-OHase Zeaxanthin Phytoene GGPP ATP PPi GBSSI 25% Amylose β-OHase 25% Amylose 75% Amylopectin SBEIIDE SBEI SSIII SSII SSI ATP PPi ADPG G1P β-OHase Lycopene ζ-carotene 15µg Lutein PSY GBSSI Zeaxanthin β-carotene Chromoplast Zeaxanthin β-carotene Lycopene ζ-carotene Phytoene GGPP β+ε-OHase α-carotene 25 µg Total Carotenoid Content β-OHase LCYB LCYB + LCYE β-OHase ZDS + CRTISO PDS + Z-ISO 15µg ADPG Amyloplast 25 µg Total Carotenoid Content GBSSI PSY 25 µg Total Carotenoid Content LCYB LCYB + LCYE ζ-carotene Lutein PSY ATP PPi G1P ADPG Phytoene GGPP α-carotene 25% Amylose β-carotene Lycopene Phytoene GGPP SBEIIDE SBEI SSIII SSII SSI GBSSI ADP LCYB LCYB + LCYE ZDS + CRTISO PDS + Z-ISO ZDS + CRTISO PDS + Z-ISO 75% Amylopectin ADP Amyloplast 75% Amylopectin SBEIIDE SBEI SSIII SSII SSI 25 µg Total Carotenoid Content Lutein β+ε-OHase α-carotene Zeaxanthin ATP PPi G1P Amyloplast 75% Amylopectin β-carotene β-OHase LCYB LCYB + LCYE ADPG G1P GBSSI 25% Amylose SBEIIDE SBEI SSIII SSII SSI GBSSI 25% Amylose Chromoplast 25 µg Total Carotenoid Content Chromoplast ATP PPi 75% Amylopectin ADP SBEIIDE SBEI SSIII SSII SSI LCYB LCYB + LCYE ADP ADPG 15µg β+ε-OHase 75% Amylopectin SBEIIDE SBEI SSIII SSII SSI ADPG ATP PPi G1P GBSSI 25% Amylose Lycopene ζ-carotene Phytoene PSY 15µg α-carotene SBEIIDE SBEI SSIII SSII SSI ATP PPi G1P GGPP Lutein PSY 75% Amylopectin GBSSI 25% Amylose Amyloplast β-carotene Lycopene ζ-carotene Phytoene GGPP Chromoplast ADP 75% Amylopectin SBEIIDE SBEI SSIII SSII SSI ZDS + CRTISO PDS + Z-ISO β-OHase β+ε-OHase α-carotene ATP PPi G1P Amyloplast ADPG LCYB LCYB + LCYE 25 µg Total Carotenoid Content Lutein PSY SBEIIDE SBEI SSIII SSII SSI GBSSI 25% Amylose ζ-carotene Phytoene GGPP ADP 75% Amylopectin SBEIIDE SBEI SSIII SSII SSI ATP PPi G1P
- 23. pVA and Starch: Data integrity Functional Genetic for high quality cassava: pVA and Starch synthase pathways Three years evaluation 2011, 2012 and 2013 Total Carotenoid Content (HPLC method) Carotenoid Synthase Pathway 30 25 µg Total Carotenoid Content 25 β-OHase Zeaxanthin ZDS + CRTISO PDS + Z-ISO Lycopene ζ-carotene Phytoene PSY 15 10 Lutein 15µg y = 1.0007x + 1.7402 R² = 0.821 5 β+ε-OHase α-carotene 0 0 5 10 15 20 25 2011 Chromoplast Total Carotenoid Content (HPLC method) Linear (Total Carotenoid Content (HPLC method)) β-carotene content (HPLC method) 16 14 12 10 2012 GGPP 20 β-carotene 2012 LCYB LCYB + LCYE 8 6 y = 1.0899x - 0.0065 R² = 0.8194 4 2 0 0 2 4 6 8 10 12 2011 b-carotene content (HPLC method) Linear (b-carotene content (HPLC method)) 14
- 24. Carotenoid Synthase Pathway β-OHase Zeaxanthin ZDS + CRTISO PDS + Z-ISO GGPP PSY β-carotene Lycopene Phytoene 15µg α-carotene β+ε-OHase 45 Chromoplast 40 0 GM3732-28 GM3736-78 GM3736-84 GM3736-29 GM3732-19 GM3732-22 GM3736-75 GM3736-40 GM3736-56 GM3736-26 GM3736-45 GM3736-37 GM3736-34 GM3732-02 GM3732-05 GM3732-34 GM3736-17 GM3736-16 GM3732-17 GM905-57 GM3736-32 GM3736-15 GM3732-18 GM3736-58 GM3736-59 GM3732-11 GM3732-08 GM3736-77 GM3736-20 GM3736-69 GM905-60 GM3732-29 GM3732-30 GM3736-66 GM3736-38 GM3736-36 GM3732-01 GM3736-76 GM3732-21 GM3736-51 GM3736-43 GM3732-16 GM3736-18 GM3736-85 GM3736-12 GM3736-44 GM3732-37 GM3732-13 GM3736-13 GM3736-02 GM3736-06 GM3736-48 GM3736-53 GM3736-54 GM3736-74 GM3732-23 GM3732-36 GM3736-72 GM3732-31 GM3736-57 GM905-52 GM3736-52 GM3736-03 GM3736-08 GM3732-25 GM3736-19 GM3736-11 GM3732-27 GM3736-23 GM3736-80 GM3732-12 GM3732-35 GM3736-05 GM3736-79 GM3732-14 GM3732-04 GM3736-63 GM3732-20 GM3732-32 GM3732-09 GM3736-24 GM3736-25 GM3736-41 GM3732-33 GM3736-73 GM3732-03 GM3736-10 GM3732-06 GM3736-22 GM3736-61 GM3736-35 GM3736-47 GM3736-21 GM3736-30 GM3736-27 GM3736-70 GM3732-07 GM3736-39 GM3732-15 GM3736-49 GM3736-81 GM3732-10 GM3736-09 GM3736-33 GM3736-50 GM3732-24 GM3736-28 GM3736-71 GM3736-64 GM3736-14 GM3736-55 GM3736-82 GM3736-42 GM3736-46 GM3736-62 GM3736-67 GM3736-01 GM3736-83 LCYB LCYB + LCYE Carotenoid Potential Functional Genetic for high quality cassava: pVA and Starch synthase pathways Carotenoid Gained pVA and Starch: The potential Three years evaluation 2011, 2012 and 2013 IPP 25 µg Total Carotenoid Content Geranylgeranyl diphosphate Phytoene synthase Phytoene ζ-carotene Phytoene desaturase Lutein Lycopene ξ-carotene desaturase Lycopene-beta-cyclase -carotene (vitamin A precursor) PTFN 2012 PTH 2012 TCH 2012 35 30 25 20 15 10 5
- 25. Carotenoid Synthase Pathway β-OHase Zeaxanthin ZDS + CRTISO PDS + Z-ISO GGPP PSY Lycopene β-carotene Phytoene 15µg α-carotene β+ε-OHase 45 Chromoplast 40 0 GM3732-28 GM3736-78 GM3736-84 GM3736-29 GM3732-19 GM3732-22 GM3736-75 GM3736-40 GM3736-56 GM3736-26 GM3736-45 GM3736-37 GM3736-34 GM3732-02 GM3732-05 GM3732-34 GM3736-17 GM3736-16 GM3732-17 GM905-57 GM3736-32 GM3736-15 GM3732-18 GM3736-58 GM3736-59 GM3732-11 GM3732-08 GM3736-77 GM3736-20 GM3736-69 GM905-60 GM3732-29 GM3732-30 GM3736-66 GM3736-38 GM3736-36 GM3732-01 GM3736-76 GM3732-21 GM3736-51 GM3736-43 GM3732-16 GM3736-18 GM3736-85 GM3736-12 GM3736-44 GM3732-37 GM3732-13 GM3736-13 GM3736-02 GM3736-06 GM3736-48 GM3736-53 GM3736-54 GM3736-74 GM3732-23 GM3732-36 GM3736-72 GM3732-31 GM3736-57 GM905-52 GM3736-52 GM3736-03 GM3736-08 GM3732-25 GM3736-19 GM3736-11 GM3732-27 GM3736-23 GM3736-80 GM3732-12 GM3732-35 GM3736-05 GM3736-79 GM3732-14 GM3732-04 GM3736-63 GM3732-20 GM3732-32 GM3732-09 GM3736-24 GM3736-25 GM3736-41 GM3732-33 GM3736-73 GM3732-03 GM3736-10 GM3732-06 GM3736-22 GM3736-61 GM3736-35 GM3736-47 GM3736-21 GM3736-30 GM3736-27 GM3736-70 GM3732-07 GM3736-39 GM3732-15 GM3736-49 GM3736-81 GM3732-10 GM3736-09 GM3736-33 GM3736-50 GM3732-24 GM3736-28 GM3736-71 GM3736-64 GM3736-14 GM3736-55 GM3736-82 GM3736-42 GM3736-46 GM3736-62 GM3736-67 GM3736-01 GM3736-83 LCYB LCYB + LCYE Carotenoid Potential Functional Genetic for high quality cassava: pVA and Starch synthase pathways Carotenoid Gained pVA and Starch: No Lycopene Three years evaluation 2011, 2012 and 2013 IPP 25 µg Total Carotenoid Content Geranylgeranyl diphosphate Phytoene synthase Phytoene ζ-carotene Phytoene desaturase Lutein Lycopene ξ-carotene desaturase -carotene (vitamin A precursor) Lycopene-beta-cyclase PTFN 2012 PTH 2012 TCH 2012 35 30 25 20 15 10 5
- 26. Carotenoid Synthase Pathway β-OHase Zeaxanthin ZDS + CRTISO PDS + Z-ISO GGPP PSY β-carotene Lycopene Phytoene 15µg α-carotene β+ε-OHase 45 Chromoplast 40 15 0 GM3732-28 GM3736-78 GM3736-84 GM3736-29 GM3732-19 GM3732-22 GM3736-75 GM3736-40 GM3736-56 GM3736-26 GM3736-45 GM3736-37 GM3736-34 GM3732-02 GM3732-05 GM3732-34 GM3736-17 GM3736-16 GM3732-17 GM905-57 GM3736-32 GM3736-15 GM3732-18 GM3736-58 GM3736-59 GM3732-11 GM3732-08 GM3736-77 GM3736-20 GM3736-69 GM905-60 GM3732-29 GM3732-30 GM3736-66 GM3736-38 GM3736-36 GM3732-01 GM3736-76 GM3732-21 GM3736-51 GM3736-43 GM3732-16 GM3736-18 GM3736-85 GM3736-12 GM3736-44 GM3732-37 GM3732-13 GM3736-13 GM3736-02 GM3736-06 GM3736-48 GM3736-53 GM3736-54 GM3736-74 GM3732-23 GM3732-36 GM3736-72 GM3732-31 GM3736-57 GM905-52 GM3736-52 GM3736-03 GM3736-08 GM3732-25 GM3736-19 GM3736-11 GM3732-27 GM3736-23 GM3736-80 GM3732-12 GM3732-35 GM3736-05 GM3736-79 GM3732-14 GM3732-04 GM3736-63 GM3732-20 GM3732-32 GM3732-09 GM3736-24 GM3736-25 GM3736-41 GM3732-33 GM3736-73 GM3732-03 GM3736-10 GM3732-06 GM3736-22 GM3736-61 GM3736-35 GM3736-47 GM3736-21 GM3736-30 GM3736-27 GM3736-70 GM3732-07 GM3736-39 GM3732-15 GM3736-49 GM3736-81 GM3732-10 GM3736-09 GM3736-33 GM3736-50 GM3732-24 GM3736-28 GM3736-71 GM3736-64 GM3736-14 GM3736-55 GM3736-82 GM3736-42 GM3736-46 GM3736-62 GM3736-67 GM3736-01 GM3736-83 LCYB LCYB + LCYE Carotenoid Potential Functional Genetic for high quality cassava: pVA and Starch synthase pathways Carotenoid Gained pVA and Starch: Unknown carotenoids Three years evaluation 2011, 2012 and 2013 IPP 25 µg Total Carotenoid Content Geranylgeranyl diphosphate Phytoene synthase Phytoene ζ-carotene Phytoene desaturase Lutein Lycopene ξ-carotene desaturase Lycopene-beta-cyclase -carotene (vitamin A precursor) PTFN 2012 PTH 2012 TCH 2012 35 30 25 20 Unknown Carotenoids 10 5
- 27. Carotenoid Synthase Pathway β-OHase Zeaxanthin ZDS + CRTISO PDS + Z-ISO GGPP PSY β-carotene Lycopene Phytoene 15µg α-carotene β+ε-OHase Chromoplast 25 0 GM3732-28 GM3736-78 GM3736-84 GM3736-29 GM3732-19 GM3732-22 GM3736-75 GM3736-40 GM3736-56 GM3736-26 GM3736-45 GM3736-37 GM3736-34 GM3732-02 GM3732-05 GM3732-34 GM3736-17 GM3736-16 GM3732-17 GM905-57 GM3736-32 GM3736-15 GM3732-18 GM3736-58 GM3736-59 GM3732-11 GM3732-08 GM3736-77 GM3736-20 GM3736-69 GM905-60 GM3732-29 GM3732-30 GM3736-66 GM3736-38 GM3736-36 GM3732-01 GM3736-76 GM3732-21 GM3736-51 GM3736-43 GM3732-16 GM3736-18 GM3736-85 GM3736-12 GM3736-44 GM3732-37 GM3732-13 GM3736-13 GM3736-02 GM3736-06 GM3736-48 GM3736-53 GM3736-54 GM3736-74 GM3732-23 GM3732-36 GM3736-72 GM3732-31 GM3736-57 GM905-52 GM3736-52 GM3736-03 GM3736-08 GM3732-25 GM3736-19 GM3736-11 GM3732-27 GM3736-23 GM3736-80 GM3732-12 GM3732-35 GM3736-05 GM3736-79 GM3732-14 GM3732-04 GM3736-63 GM3732-20 GM3732-32 GM3732-09 GM3736-24 GM3736-25 GM3736-41 GM3732-33 GM3736-73 GM3732-03 GM3736-10 GM3732-06 GM3736-22 GM3736-61 GM3736-35 GM3736-47 GM3736-21 GM3736-30 GM3736-27 GM3736-70 GM3732-07 GM3736-39 GM3732-15 GM3736-49 GM3736-81 GM3732-10 GM3736-09 GM3736-33 GM3736-50 GM3732-24 GM3736-28 GM3736-71 GM3736-64 GM3736-14 GM3736-55 GM3736-82 GM3736-42 GM3736-46 GM3736-62 GM3736-67 GM3736-01 GM3736-83 LCYB LCYB + LCYE Carotenoid Potential Functional Genetic for high quality cassava: pVA and Starch synthase pathways Carotenoid Gained pVA and Starch: DMC do not matter Carotenoids vs Dry Matter Content (DMC) IPP Geranylgeranyl diphosphate Gains from 2010/12 to 2013 From 25ug to 29ug in TCC From 15ug to 22ug in TBC 25 µg Total Carotenoid Content Phytoene synthase Phytoene ζ-carotene Phytoene desaturase Lutein Lycopene ξ-carotene desaturase Lycopene-beta-cyclase (vitamin A precursor) Dry Matter Content (2012) -carotene 50 45 40 35 30 20 High β-carotene has no penalty on DMC 15 10 5
- 28. pVA and Starch: Novel starches Functional Genetic for high quality cassava: pVA and Starch synthase pathways Waxy and small-granule cassava Easy hydrolysis good for bioethanol and bioplastic production 75% Amylopectin DE SBEII SBEI SSIII SSII SSI X Starch Synthase Pathway ATP PPi 75% Amylopectin G1P DE SBEII SBEI SSIII SSII SSI ADPG ADP ATP PPi G1P GBSSI ADPG 25% Amylose ADP GBSSI 25% Amylose Amyloplast Amyloplast
- 29. pVA and Starch: Novel starches Functional Genetic for high quality cassava: pVA and Starch synthase pathways Waxy and small-granule cassava 75% Amylopectin DE SBEII SBEI SSIII SSII SSI Starch Synthase Pathway ATP PPi 75% Amylopectin G1P DE SBEII SBEI SSIII SSII SSI ADPG ADP XGBSSI ATP PPi G1P ADPG 25% Amylose ADP GBSSI 25% Amylose Amyloplast Amyloplast
- 30. pVA and Starch: Novel starches WAXY Functional Genetic for high quality cassava: pVA and Starch synthase pathways Waxy and small-granule cassava Starch Synthase Pathway 75% Amylopectin DE SBEII SBEI SSIII SSII SSI ATP PPi G1P ADPG ADP GBSSI 25% Amylose Amyloplast • GBSSI in cassava is ~3400 bp • 2 to 3 allelic versions • Among 7 different sources of the mutation span 126 SNPs • 3 are diagnostic of the trait
- 31. pVA and Starch: Team and Founder Functional Genetic for high quality cassava: pVA and Starch synthase pathways Carotenoid Synthase Pathway 25 µg Total Carotenoid Content β-OHase Zeaxanthin LCYB LCYB + LCYE ZDS + CRTISO PDS + Z-ISO β-carotene Lycopene ζ-carotene Phytoene GGPP Lutein PSY 15µg β+ε-OHase α-carotene Chromoplast Starch Synthase Pathway 75% Amylopectin DE SBEII SBEI SSIII SSII SSI ATP PPi G1P ADPG ADP GBSSI 25% Amylose Amyloplast
- 32. Whitefly research Whitefly control: a durable solution to cassava-disease pandemics (Africa and Asia) Whitefly DNA barcoding Whitefly resistance 6 new species molecular identification A B Bemisia tabaci biotypeB KF059959 CIAT 99 D A Bemisia tabaci biotypeA KF059958 CIAT 96 C Asia / Asia-Minor Bemisia tabaci JN410738 100 CMC-40 Bemisia tabaci JN410741 30 New World G-I 99 81 Bemisia tabaci AY521259 Aleurolobus marlatti JQ340188 Asia Siphoninus philyreae JQ340199 Asia / Asia-Minor Bemisia tabaci JN410738 Bemisia tabaci biotypeB KF059959 CIAT 100 A Bemisia tabaci biotypeA KF059958 CIAT 96 99 30 New World 88 C Aleurothrachelus socialis KF059953 CIAT 89 88 New World 93 E New World Trialeurodes variabilis KF059962 CIAT Aleuroclava sp JQ340187 93 Asia Dialeurodes hongkongensis JQ340195 New World Trialeurodes vaporiariorum KF192508 CIAT F 100 Trialeurodes vaporariorum AY521265 G Aleurodicus dugessi AY521251 46 99 Lecanoideus floccissimus KF059960 CIAT H Aleurodicus dispersus KF059954 CIAT 95 99 Aleurodicus dispersus EU581838 I 75 Aleurodicus dispersus EU581837 New World G-V Asia Drosophila melanogaster ADW61333 G-VI Drosophila melanogaster ADW61439 100 Drosophila melanogaster AGE13862 0.10 0.05 Aleuronudus melzeri KF059955 CIAT New World G Aleurodicus dugessi AY521251 46 99 Lecanoideus floccissimus KF059960 CIAT H Aleurodicus dispersus KF059954 CIAT 95 99 Aleurodicus dispersus EU581838 I 75 Aleurodicus dispersus EU581837 0.00 Drosophila melanogaster ADW61439 100 Drosophila melanogaster ADW61333 Drosophila melanogaster AGE13862 0.20 0.15 0.10 0.05 0.00 G-VI Aleurodicinae Aleuronudus melzeri KF059955 CIAT F 100 Trialeurodes vaporariorum AY521265 E Trialeurodes variabilis KF059962 CIAT 99 Trialeurodes vaporiariorum KF192508 CIAT 99 Aleurodicinae Dialeurodes citri JQ340193 98 G-III Dialeuropora JQ340197 73 0.15 Asia Dialeurodes hongkongensis JQ340195 D Aleurothrixus floccosus KF059956 CIAT 68 0.20 Dialeurodes citri JQ340193 98 G-II Asia Aleurotrachelus trachoides KF059957 CIAT 98 Dialeuropora JQ340197 73 Crenidorsum micheliae JQ340190 97 G-II Aleuroclava sp JQ340187 B G-IV Aleyrodinae 45 New World D Aleurothrixus floccosus KF059956 CIAT Asia Aleurocanthus inceratus HM150621 Aleurocanthus spiniferus JQ340176 Aleurothrachelus socialis KF059953 CIAT 89 Siphoninus philyreae JQ340199 99 C Aleurotrachelus trachoides KF059957 CIAT 98 68 Bemisia tabaci AY521259 Aleurolobus marlatti JQ340188 Crenidorsum micheliae JQ340190 97 G-IV Bemisia tabaci JN410741 G-I 99 81 45 B Asia G-V Whitefly molecular identification Aleurocanthus spiniferus JQ340176 G-III Aleyrodinae MEcu 72 Aleurocanthus inceratus HM150621 99 Asia
- 33. Whitefly research Whitefly control: a durable solution to cassava-disease pandemics (Africa and Asia) Whitefly resistance A B CMC-40 C D MEcu 72 Whitefly molecular identification Bemisia tabaci JN410741 Asia / Asia-Minor Bemisia tabaci JN410738 Bemisia tabaci biotypeB KF059959 CIAT A Bemisia tabaci biotypeA KF059958 CIAT 96 99 30 New World G-I 99 81 100 Bemisia tabaci AY521259 Aleurolobus marlatti JQ340188 Asia Siphoninus philyreae JQ340199 Aleurocanthus inceratus HM150621 45 B Asia Crenidorsum micheliae JQ340190 97 C Aleurotrachelus trachoides KF059957 CIAT 98 Aleurothrachelus socialis KF059953 CIAT 89 88 New World G-II Aleurocanthus spiniferus JQ340176 D Aleurothrixus floccosus KF059956 CIAT Aleuroclava sp JQ340187 Dialeurodes citri JQ340193 98 93 G-III Dialeuropora JQ340197 73 Asia Dialeurodes hongkongensis JQ340195 E New World Trialeurodes variabilis KF059962 CIAT Trialeurodes vaporiariorum KF192508 CIAT 99 G-IV 68 New World G-V Aleyrodinae 99 F 100 Trialeurodes vaporariorum AY521265 Aleurodicinae Aleuronudus melzeri KF059955 CIAT G Aleurodicus dugessi AY521251 46 99 Lecanoideus floccissimus KF059960 CIAT H Aleurodicus dispersus KF059954 CIAT 95 99 Aleurodicus dispersus EU581838 I 75 Aleurodicus dispersus EU581837 100 Drosophila melanogaster ADW61333 Drosophila melanogaster AGE13862 0.20 0.15 0.10 0.05 0.00 G-VI Drosophila melanogaster ADW61439 Asia Whitefly DNA species identification assay
- 35. THANK YOU