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Samir V . Sawant Principal Scientist CSIR-National Botanical Research Institute Rana Prarap Marg Lucknow-226001

Genomics of Gossypium spp . for Development of Genetic Markers and Discovery of Genes Related to Fiber and Drought Traits. Samir V . Sawant Principal Scientist CSIR-National Botanical Research Institute Rana Prarap Marg Lucknow-226001. Synopsis of Presentation:.

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Samir V . Sawant Principal Scientist CSIR-National Botanical Research Institute Rana Prarap Marg Lucknow-226001

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  1. Genomics of Gossypium spp. for Development of Genetic Markers and Discovery of Genes Related to Fiber and Drought Traits Samir V. Sawant Principal Scientist CSIR-National Botanical Research Institute RanaPrarapMarg Lucknow-226001

  2. Synopsis of Presentation: Large Scale Genomic Resource Development in Cotton. Genes Underlying Drought Tolerance & Fiber Quality Traits.

  3. Large Scale Genomic Resource Development in Cotton though Sequencing of HMPR libraries

  4. Selection of Six Diverse Germplasms of G. hirsutum (based on AFLP genetic diversity) Jena et al. (2011) Crop & Pasture Science 62:859-75

  5. Genic-Enrichment by Methylation Sensitive Restriction Digestion In-sensitive Sensitive M BstBIHpaII ClaIHindIIIEcoRV Uncut DNA Enriched DNA Digestion of genomic DNA with different enzymes for methylation pattern Rai et al. (2013) Plant Biotechnology J.

  6. Reads Generated for Various Genic-enriched Cotton Genotypes

  7. Genotype wise Comparison of Genic-enriched reads using gsMapper v2.5.3

  8. Enzyme wise comparison of Genic-enriched reads using gsMapper v2.5.3 Enzyme wise comparison of HMPR enriched reads

  9. De novo Assembly using Newbler v2.5.3 Assembler

  10. Gene Prediction and Annotations Gene Prediction AUGUSTUS 90294 GenScan 125422 GlimmmerHMM 97533 Reciprocal Blast Common gene models (present in any of two or more prediction tools) 93363 Full length genes 21399 Annotation NCBI nr Total hits: 63950 Unique: 38645 TAIR 10 Total hits: 52838 Unique: 16956 Cotton EST Total hits: 45054 Unique: 19513

  11. Similarity of Predicted Gene Models with Other Plant Genomes V. vinifera R. communis A. thaliana G. raimondii

  12. qRT PCR Validation of 12 Randomly Selected Predicted Gene Models in G. hirsutum Y- axis: Fold Expression in Fiber and Root as compared to Leaf tissues

  13. Identification of Transcription Factor Encoding Genes

  14. qRT PCR Validation of 12 Randomly Selected Predicted Transcription factor encoding Gene Models in G. hirsutum Y- axis: Fold Expression in Fiber and Root as compared to Leaf tissues

  15. Genome-wide SNP Discovery in G. hirsutum SNP discovery using Newbler v2.5.3 Assembler Assembly of individual germplasms Assembly (Newbler v2.5.3) Pooling contigs from each germplasm Assembly (Newbler v2.5.3) contigs Super contigs AutoSNP Output of AutoSNP Using customized program Filtered out False SNP Detected SNP

  16. Strategy for SNP Discovery in G. hirsutum G. hirsutum genotype-1 G. hirsutum genotype-2 Assembly using gsAssembler v2.5.3 (40 bp overlap with 97% identity) autoSNP v2.0 for contigs with minimum six reads (≥3 reads from each genotype) A A G A A G C A A C A C A T C A T C T C C T C C × × Allelic SNP (Taken) Non-Allelic SNP (Discarded)

  17. Genome-wide SNP Discovery in G. hirsutum… Cultivars SNP summary Sequence alignment JKC 703 LRA 5166 LRA 5166 JKC 725 JKC 725 JKC 725 JKC 770 JKC 770 JKC 770 JKC 770

  18. Genome-wide SNP Discovery in G. hirsutum… v Details of SNP discovery Distribution of identified SNPs UTRs, 4446 Intronic, 4518

  19. TC MCU5 Validation of Identified SNPs in G. hirsutum TC JKC 770 SNPs used for Validation : 30 Germplasms used : 6 SNPs Detected : 30 TC JKC 725 TC JKC 737 TC JKC 703 LRA 5166 TC

  20. SSRs identification and Novelty comparison against Cotton Marker Database SSRs distribution on the basis of Motifs SSR novelty analysis 47,093 Novel SSRs Number of SSRs Unit size of different repeat type Unmatched whole sequence wise Total SSRs Identified 1,48,930 Unmatched primer sequence wise SSRs Successful in designing primers 56,142 Unmatched flanking sequence wise Novel SSRs developed 47,093

  21. Validation of Identified SSRs in G. hirsutum 291/297/300 bp JKC 703 SSRs used for Validation : 40 Germplasms used : 12 Polymorphic SSRs : 6 % Polymorphism : 15 291/300 bp JKC 770 291/300 bp LRA 5166 291/297 bp JKC 725 291/297 bp MCU 5 291/297 bp JKC 737

  22. Distribution of G. hirsutum SSRs and SNPs containing sequences on G. raimondii reference genome SSRs SNPs G. raimondii (JGI) G. raimondii (Chinese draft)

  23. miRNA Novel to Gossypium miR-3696 miR-1713 miR-2112 miR-165 miR-4343 miR-437 miR-4371 Novel miRNAs miR-5023 miR-477 miR-5065 miR-3963 miR-536 miR-5555 miR-950 miR-1070 miR-2675 miR-3522

  24. Promoters and Cis Regulatory Elements Size Distribution of Identified Promoters No. of Promoters Size in bases Elongation (28) Initiation (184) Sec. Cell Wall Synthesis (110)

  25. Genomic Resources Developed for G. hirsutum An Overview v Assembled Sequences 4095128 AssembledBases 1272 Mb GC Content 37.76 % Novel SNPs 66364 Repetitive content 12.16 % Novel SSRs 47093 Gene Models 93363 TF’s 1093 Promoters 3135 Full length genes 21399 Rai et al. (2013) Plant Biotechnology J.

  26. Development & Characterization of gSSRs and eSSRs in Diploid Cotton (G. herbaceum) Total SSRs from G. herbaceum UPGMA tree of 15 genotypes of G. herbaceum based on Nei’s genetic distance using 200 SSRs 263 gSSRs 1970 eSSRs Drought Transcriptome Sequencing Repeat Enriched Genomic Libraries Cross-species transferability of G. herbaceumderived gSSRs and eSSRs SSR “NBRI_gB010” among four species of cotton Jena et al. (2012) Theoretical Applied Genetics 124 (3):565-76

  27. Development of molecular markers from Indian genotypes of two Gossypium L. species G. hirsutum G. herbaceum Vagad (Drought tolerant) RAHS-14 (Drought Sensitive) JKC 703 (superior in fibre quality) JKC 777 (inferior in fibre quality) GujCot (Drought tolerant) RAHSIPS-187 (Drought Sensitive) 2608 SNPs 1440 SSRs 10,947 SNPs 50bp Flanking NBRI SNPs SSR Sequence Public Domain SNPs NBRI SNPs Public Domain SNPs 111 20 2 2 38,780 1,847 334 307 15 0 0 10,947 206 Primers 334 Novel SSRs 1,847 Novel SNPs 10,947 Novel SNPs Srivastavaet al., Journal Plant Breeding doi:10.1111/pbr.12087 (In Press)

  28. Microarray Based Single Feature Polymorphisms (SFPs) in Gossypium hirsutum Superior fiber quality Inferior fiber quality In Silicoanalysis of 37,473 SFPs in sixcrosses Biological replicate 1, 2,3 Biological replicate 1, 2,3 RNA extraction/microarray hybridization RMA background correction “770-1,2,3.CEL” “703-1,2,3.CEL” Validation of SFPs in two germplasm (JKC 703 x JKC 770) “725-1,2,3.CEL” “737-1,2,3.CEL” No. of Selected SFPs 224 No. of SNPs found 122 No. of indels found 10 “783-1,2,3.CEL” Further analysis for SFPs Srivastava et al. (2012) Communicated

  29. SSRs/SNPs/SFPs development from Cotton at NBRI NBRI COTTON MARKERS A-genome derived SSRs (genomic & expressed) 2,233 Total SSRs 59,805 AD-genome derived SSRs (Genic enrichment) 56,142 AD-Genome derived SSRs (Transcriptome sequencing) 1440 A-Genome derived SNPs (Transcriptome sequencing) 592 Total SNPs 69,768 AD-genome derived SNPs (Genic enrichment) 66,444 AD-genome derived SNPs (Transcriptome sequencing) 2,600 AD-genome derived SFPs (Microarray based) 132 1,29,573 Total Novel Markers

  30. COTTON SNP CHIP (Affymetrix’s Axiom®myDesign Cotton Array) (CSIR-NBRI) Axiom™ myDesign™ Array: Targeted genotyping, tailored for our study A streamlined assay: Total genomic DNA (200 ng) is amplified and randomly fragmented into 25 to 125 base pair (bp) fragments. These fragments are purified, re-suspended, and hybridized to Axiom Genome-Wide and myDesign Array Plates. Following hybridization, the bound target is washed under stringent conditions to remove non-specific background to minimize background noise caused by random ligation events. Each polymorphic nucleotide is queried via a multi-color ligation event carried out on the array surface. After ligation, the arrays are stained and imaged on the Gene Titan MC Instrument. Axiom myDesign TG Array Plates enable us to: Easily select relevant SNPs from our SNP database Creating panels of 500,000 markers per sample • Targeting 50,000 SNPs for Genotyping with Mapping Population

  31. Deployment of COTTON SNP CHIP on Mapping Populations • CICR, Nagpur: • a. H X H RIL population (Fiber Traits) • b. A X He RIL population (Mapping and Fiber Traits) • 2. UAS, Dharwad: • a. H X B RIL population (Fiber Traits) • b. Core Collection (Association Mapping) • 3. TNAU, Coimbatore: • a. H X H RIL population (Fiber Traits) • b. H X H RIL population (Sap sucking pests)

  32. NBRI’s Cotton Database A Webpage for Cotton Resources

  33. II. Genes Underlying Drought Tolerance & Fiber Quality Traits

  34. Screening of Cotton Genotypes for Drought Tolerance and Sensitivity Screening of G. herbaceum genotypes on different concentrations of mannitol Effect of drought on tolerant and sensitive genotype Tolerant genotype (Vagad) Drought sensitive Continuous watering Drought Tolerant Continuous watering Drought sensitive 1 week alternate watering Drought Tolerant 1 week alternate watering Sensitive genotype (RAHS-14) Ranjan et.al., BMC Genomics (2012) 13:94

  35. Physiological Parameters in Response to Drought in Vagad and RAHS- 14 • Properties of Vagad • Reduced stomatal conductance (gs) • Decreased transpiration rate (E) • Reduced water potential (WP) • Higher realtive water content (RWC) • Leading to better water use efficiency (WUE). • Vagad has inherent ability to sense the drought at much early stage and respond to it in much efficiently. Ranjan A et.al., BMC genomics 2012 March RAHS-14 Vagad

  36. Transcriptional profiling during drought and water condition in Leaf tissue of Vagad and RAHS-14 • Microarray data Pyrosequencing data Ranjan A et.al., BMC genomics 2012 March

  37. Genome wide gene expression profiling of leaf tissue of Vagad and RAHS-14 Vagad • Propanoid pathway • Pigment biosynthesis • Polyketide biosynthesis • Responses to various abiotic stresses • Secondary metabolite pathways RAHS-14 • Ethylene responsive factor • WRKY • Programmed cell death • Senescence • Lipid metabolism Ranjan A et.al., BMC genomics 2012 March

  38. Comparative root Transcriptome Analysis of Drought Tolerant and Sensitive Genotypes of G. herbaceum Drought tolerant Drought sensitive Pyrosequencing data Root architectures • Microarray data Ranjan A et.al., BMC genomics 2012 November

  39. Functional enrichment of genes of root tissue in drought tolerant and sensitive genotypes Tolerant genotype Sensitive genotype Regulation of Transcription factors (TFs) under drought stress Ranjan A et.al., BMC genomics 2012 November

  40. Differentially expressed genes analyzed by Genevestigator in mapping the specific expression of genes in different root zones Ranjan et.al., BMC Genomics (2012) 13: 680

  41. Selection of Candidate Gene for Studying the Abiotic Response Identification of Transcription Activator (TA) from Cotton Transcriptome of root tissue Expression of TA (TL-tolerant leaf, TR-tolerant root, SL-sensitive leaf, SR-sensitive root)

  42. Increased tolerance to drought and salt stress and better root development in tobacco over expressing GheTA GheTA WT WT GheTA WT GheTA GheTA WT control control 50 mMNaCl 50 mM Mannitol 75 mMNaCl 150 mM Mannitol 100 mMNaCl 200 mM Mannitol 150 mMNaCl 250 mM Mannitol

  43. Abiotic stress tolerance of the GheTA over-expressing tobacco transgenic plants by leaf disk assay WT GheTA WT GheTA Control 5 % PEG 10 % PEG 250 mM Mannitol 500 mM Mannitol 100 mMNaCl 150 mMNaCl

  44. Over-Expression of GheTAleads to increased root biomass and better WUE in cotton transgenic plants Wild type Cotton Transgenic Carbon Isotope Discrimination ratio shows higher water use efficiency (WUE) of cotton TA transgenic plants

  45. Expressional Reprogramming During Fiber Development In ContrastingGenotypesof G. hirsutum Fiber quality of genotypes Fibercellulose content Fiber lignin content Nigam et al. (2013) Communicated

  46. Method used for Analyzing Microarray Data from Contrasting Cotton Genotypes Two way ANOVA study • Microarray data analysis

  47. Genotype significant genes Singular Enrichment Analysis (SEA) DPA significant genes Interaction significant genes

  48. MapMan Bins Cluster Analysis Inferior genotypes Superior genotypes A B 0DPA 6DPA 9DPA 12DPA 19DPA 0DPA 6DPA 9DPA 12DPA 19DPA 25DPA Cluster-1 Cluster-4 6DPA 0DPA 9DPA 12DPA 19DPA 25DPA 6DPA 0DPA 12DPA 25DPA 9DPA 19DPA Cluster-2 Cluster-5 0DPA 6DPA 6DPA 0DPA 9DPA 12DPA 19DPA 25DPA 9DPA 19DPA 12DPA Cluster-6 Cluster-3

  49. Enrichment of Transcription factors

  50. 25 DPA Fiber Transcriptome: Assembly and Annotations de novo and merged assembly Annotation of unigenes

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