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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.

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Presentation on theme: "Samir V. Sawant Principal Scientist CSIR-National Botanical Research Institute Rana Prarap Marg Lucknow-226001 Genomics of Gossypium spp. for Development."— Presentation transcript:

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

2 Synopsis of Presentation: I.Large Scale Genomic Resource Development in Cotton. II.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) Genotypes Species Source of Collection JKC 703 G. hirsutum JK Agri., Hyderabad, Andhra Pradesh JKC 725 G. hirsutum JK Agri., Hyderabad, Andhra Pradesh JKC 737 G. hirsutum JK Agri., Hyderabad, Andhra Pradesh JKC 770 G. hirsutum JK Agri., Hyderabad, Andhra Pradesh LRA 5166 G. hirsutum TNAU, Coimbatore, Tamilnadu MCU5 G. hirsutum UASD, Dharwad, Karnataka Jena et al. (2011) Crop & Pasture Science 62:859-75

5 Genic-Enrichment by Methylation Sensitive Restriction Digestion M BstB I Hpa II Cla I Hind III Eco RV Digestion of genomic DNA with different enzymes for methylation pattern Uncut DNA Enriched DNA Sensitive In-sensitive Rai et al. (2013) Plant Biotechnology J.

6 GermplasmsEnzymes used Total Reads (in millions) Total Bases (in Mb) JKC 703 HpaII ClaI JKC 725 HpaII ClaI JKC 737 HpaII ClaI JKC 770 HpaII ClaI MCU5 HpaII ClaI LRA 5166 HpaII ClaI Total Reads Generated for Various Genic-enriched Cotton Genotypes

7 Germplams JKC 725JKC 737JKC 770MCU5LRA 5166 % of mapped ReadsBasesReadsBasesReadsBasesReadsBasesReadsBases JKC JKC JKC JKC MCU Genotype wise Comparison of Genic-enriched reads using gsMapper v2.5.3

8 Germplams% reads mapped% bases mapped JKC JKC JKC JKC MCU LRA Enzyme wise comparison of HMPR enriched reads Enzyme wise comparison of Genic-enriched reads using gsMapper v2.5.3

9 Parameters Cotton Genotypes JKC 703JKC 725JKC 737JKC 770MCU5LRA 5166 Super assembly All Contigs (>100 bp)58,14261,86254,73153,41927,95263, Singletons (millions) Total bases (Mb) Large contigs (>500 bp)21, ,96017,6578,66325, Largest contig size (Kb) Avg. contig size (bases) N50 contig size (bases) Q40 plus bases De novo Assembly using Newbler v2.5.3 Assembler

10 Gene Prediction and Annotations AUGUSTUS GenScan GlimmmerHMM Common gene models (present in any of two or more prediction tools) NCBI nr Total hits: Unique:38645 TAIR 10 Total hits: Unique: Cotton EST Total hits: Unique: Gene Prediction Full length genes Reciprocal Blast Annotation

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

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

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

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

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

18 Distribution of identified SNPs Details of SNP discovery Total identified SNPs4,22,617 True SNPs called75,714 Non-redundant SNPs66,444 Novel SNPs66,364 Annotated Exonic SNPs Synonymous2604 Non-synonymous SNPs6506 Genome-wide SNP Discovery in G. hirsutum… UTRs, 4446 Intronic, 4518 v

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

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

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

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

23 miRNAs in Gossypium (on the basis of miRBase) Total miRNAs identified78 miRNA families identified42 miRNA novel to G. hirsutum17 Novel miRNAs Novel miRNAs miR-1713 miR-2112 miR-2675 miR-3522 miR-3696 miR-165 miR-437 miR-477 miR-536 miR-950 miR-1070 miR-4343 miR-4371 miR-5023 miR-5065 miR-5555 miR-3963 miRNA Novel to Gossypium

24 Promoters identified24839 ≥ 1000 bp826 ≥ 500 bp3135 Fiber developmental stage specific Promoters Initiation184 Elongation28 Secondary cell wall 110 Size Distribution of Identified Promoters Promoters and Cis Regulatory Elements Size in bases No. of Promoters Initiation (184) Elongation (28) Sec. Cell Wall Synthesis (110)

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

26 Development & Characterization of gSSRs and eSSRs in Diploid Cotton (G. herbaceum) Jena et al. (2012) Theoretical Applied Genetics 124 (3): Cross-species transferability of G. herbaceum derived gSSRs and eSSRs UPGMA tree of 15 genotypes of G. herbaceum based on Nei’s genetic distance using 200 SSRs Total SSRs from G. herbaceum 263 gSSRs1970 eSSRs Repeat Enriched Genomic Libraries Drought Transcriptome Sequencing SSR “NBRI_gB010” among four species of cotton

27 10,947 SNPs 1440 SSRs 2608 SNPs SSR Sequence 50bp Flanking Primers 1, ,780 NBRI SNPs Public Domain SNPs NBRI SNPs Public Domain SNPs 10, Novel SSRs1,847 Novel SNPs10,947 Novel SNPs Srivastava et al., Journal Plant Breeding doi: /pbr (In Press) G. hirsutumG. herbaceum Development of molecular markers from Indian genotypes of two Gossypium L. species JKC 703 (superior in fibre quality) JKC 777 (inferior in fibre quality) Vagad (Drought tolerant) RAHS-14 (Drought Sensitive) GujCot (Drought tolerant) RAHSIPS-187 (Drought Sensitive)

28 (JKC725) (JKC770) (JKC703) (JKC737) (JKC783) Biological replicate 1, 2,3 RNA extraction/microarray hybridization RMA background correction “770-1,2,3.CEL” “725-1,2,3.CEL” “703-1,2,3.CEL” “737-1,2,3.CEL” “783-1,2,3.CEL” Further analysis for SFPs In Silico analysis of 37,473 SFPs in six crosses Validation of SFPs in two germplasm (JKC 703 x JKC 770) No. of Selected SFPs 224 No. of SNPs found 122 No. of indels found 10 Microarray Based Single Feature Polymorphisms (SFPs) in Gossypium hirsutum Srivastava et al. (2012) Communicated Inferior fiber quality Superior fiber quality

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

30 COTTON SNP CHIP (Affymetrix’s Axiom® myDesign Cotton Array) (CSIR-NBRI) COTTON SNP CHIP (Affymetrix’s Axiom® myDesign Cotton Array) (CSIR-NBRI) Targeting 50,000 SNPs for Genotyping with Mapping Population Axiom myDesign TG Array Plates enable us to: Easily select relevant SNPs from our SNP database Creating panels of 500,000 markers per sample 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.

31 Deployment of COTTON SNP CHIP on Mapping Populations Deployment of COTTON SNP CHIP on Mapping Populations 1.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 Mannitol percentage AccessionsControl2%4%6%8% Vagad Guj cot RAHS RAHS-IPS H AH-7GP AH AH RAS DB RAHS JYLEHAR GH-18-2LC RAHS Screening of G. herbaceum genotypes on different concentrations of mannitol Tolerant genotype (Vagad) Sensitive genotype (RAHS-14) Effect of drought on tolerant and sensitive genotype Drought sensitive Continuous watering Drought sensitive 1 week alternate watering Drought Tolerant Continuous watering Drought Tolerant 1 week alternate watering Screening of Cotton Genotypes for Drought Tolerance and Sensitivity Ranjan et.al., BMC Genomics (2012) 13:94

35 RAHS-14 Vagad 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. Physiological Parameters in Response to Drought in Vagad and RAHS- 14 Ranjan A et.al., BMC genomics 2012 March

36 Transcriptional profiling during drought and water condition in Leaf tissue of Vagad and RAHS-14 Parameters Vagad library RAHS-14 library Total reads (overlap size of 100 bp and 96% identity) a Total contigs (100 bp or greater) b Singletone Exemplar Average length of contigs350 bp180 bp Number of contigs with greater than 500 bp c Number of genes with significant hit in NCBI NR database d Number of genes with significant hit in cotton EST database Pyrosequencing data Microarray data Genotypes Differentially up regulated genes (Fold change ≥ 2) Vagad water656 RAHS- 14 water535 Vagad drought430 RAHS- 14 drought411 Ranjan A et.al., BMC genomics 2012 March

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

38 Comparative root Transcriptome Analysis of Drought Tolerant and Sensitive Genotypes of G. herbaceum Drought tolerantDrought sensitive ReadsBasesContigsSingleton Av. Contig length Av. S. length GujCot-2155, 62013, 020, 1401,28130, bp237.8bp RAHS-IPS , 30811, 199, , bp228.6bp Superconti gs 1, 04, ,219, 3472, 66450, bp231.7bp Pyrosequencing data Root architectures Genotypes Differentially up regulated genes (Fold change ≥ 2) Vagad water165 RAHS- 14 water156 Vagad drought256 RAHS- 14 drought538 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 genotypeSensitive 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 Library nameTLTRSLSR Number of Transcript (tpm)07200 Identification of Transcription Activator (TA) from Cotton Transcriptome of root tissue (TL-tolerant leaf, TR-tolerant root, SL-sensitive leaf, SR-sensitive root) Expression of TA

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

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

44 Over-Expression of GheTA leads 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 Fiber Quality Parameters Superior genotypesInferior genotypes JKC 725JKC 777JKC 703JKC 737JKC % span length (mm) Fiber strength (g/tex) Fineness (micronaire) Fiber quality of genotypes Fiber cellulose content Fiber lignin content Expressional Reprogramming During Fiber Development In Contrasting Genotypes of G. hirsutum Nigam et al. (2013) Communicated

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

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

48 MapMan Bins Cluster Analysis Cluster-1 Cluster-3 Cluster-2 0DPA9DPA12DPA19DPA25DPA 6DPA 0DPA9DPA12DPA19DPA25DPA 6DPA Cluster-4 0DPA12DPA25DPA 6DPA 12DPA19DPA25DPA 6DPA 0DPA 9DPA12DPA19DPA 6DPA 9DPA19DPA 6DPA Cluster-6 0DPA9DPA 19DPA 0DPA 12DPA Cluster-5 AB Superior genotypes Inferior genotypes

49 Enrichment of Transcription factors

50 Parameters Merged assembly of both genotypes JKC 703JKC 777 Total reads generated Total bases generated (Mb) Average read size (bp) High Quality reads used in assembly All Contigs (>100 bp) Singletons Total bases after assembly (MB) Large contigs (>500 bp) Largest contig size (bp) Average contig size (bp) N50 contig size a (bp) Aligned Reads (%) Aligned Bases (%) Inferred read error b (%) Q40 plus bases c (%) de novo and merged assembly Parameters Genotypes Merged assembly of both genotypes JKC 703JKC 777 Total unigenes a Hits in 'NCBI nr' database Hits in 'tair9' database33,35937, Hits in ESTScan ,852 Differential unigenes Annotation of unigenes 25 DPA Fiber Transcriptome: Assembly and Annotations

51 R 2 =0.68, p-value = Correlation between Cotton Fiber Microarray and Transcriptome Sequencing

52 SUPERIOR GENOTYPE INFERIOR GENOTYPE Continued Barssinosteroid Signalling JA Auxin BES1DET2 Phospholipases Pat5, Pat6 Cell wall Enzymes PG,PAE,PME,PMEI mRNA and Protein degradation Ubiquitin ligase, Proteasome, Splisosome BIN2 Transporter Machinery ABC transporter Transcription factors HSPs, Ca ++ /CAM1 PRF5 SPL3 MEE 59 Oxiplipins ALDH APY2 ABA CSEA Initiation Elongation SCW ABI3/VP1 EXL5 SPL5 AGPs LIM domain S3 BES1DET2 Barssinosteroid Targeted Gene Expression Barssinosteroid signalling BR Efficient energy source for fast elongating fiber (Oxidative Phosphorylation e.g.TCA,Glycolysis) Asp family Large number of ribosomal subunits (Better Protein synthesizing machinery ) Increased Stress Tolerance to facilitate elongation process up to its completion Increased Stress Environment within fibre cell and complete end of cell elongation process Decreased high elongation rate of fibre cells during elongation period SCW formation Starts Fibre Cells Continuative Elongation End of Cell Elongation Pectin Modification Cell wall loosening Energy Exhaust Continued Energy Providing Machinery Flavonoid Biosynthesis Barssinosteroid Targeted Gene Expression Cell death ROS Ascorbate peroxidase Glutathione -S- Transferase Ascorbate peroxidase Glutathione -S- Transferase POX H202 ROS Calcium Signalling GA H2O2 Induction of Stress Hormones signal like, Ethylene and ABA Oxidative stress Lipid peroxidation C2C2- GATA NAC WRKY AP2- EREBP HSF Hypothetical regulatory model showing over-represented genes and pathways in Superior and Inferior genotypes

53 QTL start QTL endQTL size QTL size (MB) chr size chr size (MB) % covered by QTL in a chr QTL location Total No. of genes in the QTL region Completely Mapped genes in QTL region (Our data) Total mapped gene on Chromomosome (our data) Chr Chr Chr Chr Chr Chr Chr Chr Chr Chr Chr Chr Positions of mapped differentially expressed genes on QTL Percentage of genes mapped in QTL 600/1684 = ~35% Circos Plot of all the differential genes mapped on the cotton jgi genome with all the QTL.

54 Heat map of 9 TA5 transcription factors Ghi S1_s_at (GhSPL5) microRNA156 Targeted Transcription Factor (TA5) Governs the Boll Number, Size and Lint Yield in G. hirsutum q-RT PCR measurement Gra A1_at (GhSPL13) Gra A1_at (GhSPL14)

55 GhTA5 produce transcripts that are targeted by miR156 Northern blot of miR156 & miR172 in cotton fiber

56 Overexpression Lines Wild Type Knockdown Lines Overexpression lines Knockdown lines Wild Type Number of Cotton Bolls Overexpression line Knockdown line Overexpression line Knockdown line Lint cotton weight (gm) /plant Seed cotton weight (gm) /plant Average of cotton boll per plant Phenotypic evaluation of overexpression and knockdown lines

57 Identification and characterization of fiber specific promoter in G. hirsutum (NBRI_2800) Fold Change Fiber developmental stages Expression pattern of NBRI 2800 gene in different fiber developmental stages

58 Histochemical localization of GUS expression in NBRI_2800 transgenic cotton plant

59 Acknowledgments Bioinformatics  Dr. Mehar Asif  Dr. Sumit K. Bag  Dipti Nigam  Archana Bhardwaj  Ridhi Goel  Pooman Pant Functional Genomics  Neha Pandey  Rajiv Tripathi  Vrijesh Yadav  Anshulika Sable HMPR Sequencing & Epigenetic regulation  Sunil K. Singh  Krishan M. Rai  Verandra Kumar Cotton Marker  Dr. S N Jena  Anukool Srivastava  Ravi P. Shukla Collaborators  J K Agrigenetics Tierra Seed Science  TNAU, Coimbatore CICR, Nagpur  UAS, Dharwad

60 Thank You


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