Indian Initiative for Rice Genome Sequencing (IIRGS)

Akhilesh Tyagi
Director & JC Bose National Fellow
National Institute of Plant Genome Research
New Delhi, India

India grows rice on 43 million hectares of land and has been successful in producing up to 100 million tons of rice per year. Increasing population and changing food preferences require intensive efforts to produce more rice and that too under conditions when both land and water resources are dwindling. It is, therefore, imperative to understand intrinsic yield-limiting factors at molecular level and deploy natural as well as engineered variability most effectively for sustainable rice cultivation. Rice genome sequence is indeed a useful resource for all such scientific endeavors.

After the availability of map-based sequence of rice genome in public domain, we have continued to contribute to the international efforts to help improve annotation of various genes and genetic elements, largely based on comparative genomics and molecular model based predictions. Recently, a novel prototype for manually curated database of rice proteins has been developed and the same is being implemented to integrate and digitize relevant information distributed in published literature related to rice genes and genomes.

A major effort has been undertaken on rice transcriptome analysis during reproductive development, heterosis, water-deficit conditions and in response to plant hormones to identify rice genes expressing differentially under these conditions as this can help judicious selection of targets for genetic enhancement of rice. A computation assisted manual analysis of organization, structure, phylogeny and expression of gene families for transcription factors (MADS box, zinc finger, bZIP, Homeobox, TCP, Mediator complex), epigenetic regulation components (Argonaute, RDRP, DCL, Cytosine DNA methyltransferase) and regulatory network components (SAP, CRR, AUX/IAA, GH3, SAUR, CDPK, F-box, TCS, Phototropin, RLCK, Phosphatase, Glutaredoxin, Phospholipase C, Ca transport elements, ARM) has assisted in unraveling novel monocot clade specific and rice specific genes. This has also shed light on molecular co-evolution including neo- and sub-functionalization. Genes and regulatory elements involved in genesis of various reproductive organs and responsive to extrinsic/intrinsic cues have been selected for functional characterization. OsMADS29 has been found to control embryo and endosperm development by affecting hormone homeostasis. At least five promoters conferring anther/pollen specific expression have been characterized and are being used to manipulate male sterility, with the eventual aim to utilize them for heterosis breeding. A set of genes including OsSAP, OsTOP6, OsRLCK253 and OsTCP19 have been found to alter plant response to abiotic stress including the potential to grow in water-deficit conditions. Such genes are expected to improve rice for cultivation under adverse environmental conditions.

Selected publications

1. International Rice Genome Sequencing Project (2005) The map-based sequence of the rice genome. Nature 436: 793-800.

2. Thakur JK, Jain M, Tyagi AK, Khurana JP (2005) Exogenous auxin enhances the degradation of a light down-regulated and nuclear-localized OSiIAA1, an Aux/IAA protein from rice, via proteasome. Biochim. Biophys. Acta 1736: 196-205.

3. The Rice Chromosomes 11 and 12 Sequencing Consortia (2005) The sequence of rice chromosomes 11 and 12, rich in disease resistance genes and recent gene duplication. BMC Biology 3: 20.

4. Jain M, Kaur N, Garg R, Thakur JK, Tyagi AK, Khurana JP (2006) Structure and expression analysis of early auxin-responsive Aux/IAA gene family in rice (Oryza sativa). Funct. Integr. Genomics 6: 47-59.

5. Jain M, Kaur N, Tyagi AK, Khurana JP (2006) Comprehensive analysis of auxin responsive GH3 gene family in rice (Oryza sativa). Funct. Integr. Genomics 6: 36-46.

6. Jain M, Nijhawan A, Tyagi AK, Khurana JP (2006) Validation of housekeeping genes as internal control for studying gene expression in rice by quantitative real-time PCR. Biochem. Biophys. Res. Commun. 345 (2006) 646-651.

7. Jain M, Tyagi AK, Khurana JP (2006) Genome-wide analysis, evolutionary expansion, and expression of early auxin-responsive SAUR gene family of rice (Oryza sativa). Genomics 88 (2006) 360-371.

8. Jain M, Tyagi AK, Khurana JP (2006) Molecular characterization and differential expression of cytokinin-responsive type-A response regulators in rice ( Oryza sativa). BMC Plant Biology 6: 1.

9. Jain M, Tyagi AK, Khurana JP (2006) Overexpression of putative topoisomerase 6 genes from rice confers stress tolerance in transgenic Arabidopsis plants. FEBS J. 273: 5245-5260.

10. Tyagi AK (2006) Rice genomics. Perspectives of Cytology and Genetics 12: 13-24.

11. Varshney RK, Hoisington DA, Tyagi AK (2006) Advances in cereal genomics and applications in crop breeding. Trends Biotechnol. 24: 490-497.

12. Vij S, Tyagi AK (2006) Genome-wide analysis of the stress associated protein (SAP) gene family containing A20/AN1 zinc-finger(s) in rice and their phylogenetic relationship with Arabidopsis. Mol. Genet. Genomics 276: 565-571.

13. Vij S, Gupta V, Kumar D, Vydianathan R, Raghuvanshi S, Khurana P, Khurana JP, Tyagi AK (2006) Decoding the rice genome. BioEssays 28: 421-432.

14. Agarwal P, Arora R, Ray S, Singh AK, Singh VP, Takatsuji H, Kapoor S, Tyagi AK (2007) Genome-wide identification of C2H2 zinc-finger gene family in rice and their phylogeny and expression analysis. Plant Mol. Biol. 65: 467-485.

15. Arora R, Agarwal P, Ray S, Singh A, Singh V, Tyagi AK, Kapoor S (2007) MADS-box gene family in rice: Genome-wide identification, organization and expression profiling during reproductive development and stress. BMC Genomics 8: 242.

16. Gupta V, Khurana R, Tyagi AK (2007) Promoters of two anther-specific genes confer organ-specific expression in a stage-specific manner in transgenic systems. Plant Cell Rep. 26: 1919-1931.

17. Jain M, Nijhawan A, Arora R, Agarwal P, Ray S, Sharma P, Kapoor S, Tyagi AK, Khurana JP (2007) F-box proteins in rice: genome-wide analysis, classification, temporal and spatial gene expression during panicle and seed development, and regulation by light and abiotic stress. Plant Physiol. 143: 1467-1483.

18. Jain M, Sharma P, Tyagi SB, Tyagi AK, Khurana JP (2007) Light regulation and differential tissue-specific expression of phototropin homologues from rice (Oryza sativa ssp. indica). Plant Sci. 172: 164-171.

19. Kathuria H, Giri J, Tyagi H, Tyagi AK. Advances in transgenic rice biotechnology. Crit. Rev. Plant Sci. 26: 65-103.

20. Ray S, Agarwal P, Arora R, Kapoor S, Tyagi AK (2007) Expression analysis of calcium-dependent kinase gene family during reproductive development and abiotic stress conditions in rice (Oryza sativa L. ssp. Indica). Mol. Genet. Genomics 278: 493-505.

21. Rice Annotation Project (2007) Curated genome annotation of Oryza sativa ssp. Japonica and comparative genome analysis with Arabidopsis thaliana. Genome Res. 17: 175-183.

22. Vij S, Tyagi AK (2007) Emerging trends in functional genomics of abiotic response in crop plants. Plant Biotech. Jour. 5: 361-380.

23. Jain M, Khurana P, Tyagi AK, Khurana JP (2008) Genome-wide analysis of intronless genes in rice and Arabidopsis. Funct. Integr. Genomics 8: 69-78.

24. Jain M, Tyagi AK, Khurana JP (2008) Constitutive expression of a meiotic recombination protein gene homolog, OsTOP6A1, from rice confers abiotic stress tolerance in transgenic Arabidopsis plants. Plant Cell Rep. 27: 767-778.

25. Jain M, Tyagi AK, Khurana JP (2008) Differential gene expression of rice two-component signaling elements during reproductive development and regulation by abiotic stress. Funct. Integr. Genomics 8: 175-180.

26. Jain M, Tyagi AK, Khurana JP (2008) Genome-wide identification, classification, evolutionary expansion and expression analyses of homeobox genes in rice. FEBS J. 275: 2845-2861.

27. Kapoor M, Arora R, Lama T, Nijhawan A, Khurana JP, Tyagi AK, Kapoor S (2008) Genome-wide identification, organization and phylogenetic analysis of dicer-like, argonaute and RNA-dependent RNA polymerase gene families and their expression analysis during reproductive development and stress in rice. BMC Genomics 9: 451.

28. Nizhawan A, Jain M, Tyagi AK, Khurana JP (2008) A genomic survey and gene expression analysis of basic leucine zipper (bZIP) transcription factor family in rice. Plant Physiol. 146: 333-350.

29. Rice Annotation Project (2008) The rice annotation project database (RAP-DB): 2008 update. Nucl. Acids Res 36: D1028-D1033.

30. Vij S, Tyagi AK (2008). A20/AN1 zinc-finger domain-containing proteins in plants and animals represent common elements in stress response. Funct. Integr. Genomics 8: 301-307.

31. Vij S, Giri J, Dansana P. Kapoor S & Tyagi AK (2008) The receptor-like cytoplasmic kinase (OsRLCK) gene family in rice: organization, phylogenetic relationship and expression during development and stress. Molecular Plant 1 (2008) 732-750.

32. Chaudhary N, Khurana P (2009) Vitamin E biosynthesis genes in rice: molecular characterization, expression profiling and comparative phylogenetic analysis. Plant Sci. 177: 479-492.

33. Sharma R, Singh RKM, Malik G, Deveshwar P, Tyagi AK, Kapoor S, Kapoor M (2009) Rice cytosine DNA methyltransferases--gene expression profiling during reproductive development and abiotic stress. FEBS J. 276: 6301-6311.

34. Singla B, Khurana JP, Khurana P (2009) Structural characterization and expression analysis of the SERK gene family in rice. Int. J. Plant Genomics ( doi:10.1155/2009/539402).

35. Anand S, Tyagi AK (2010) Characterization of a pollen-preferential gene OSIAGP from rice (Oryza sativa L. subspecies indica) coding for an arabinogalactan-protein homolog and analysis of its promoter activity during pollen development and pollen tube growth. Transgenic Res. 19: 385-397.

36. Chaudhary N, Nijhawan A, Khurana JP, Khurana P (2010) Carotenoid biosynthesis genes in rice: Structural analysis, genome-wide expression profiling and phylogenetic analysis. Mol. Genet. Genomics 283: 13-33.

37. Garg R, Jhanwar S, Tyagi AK, Jain M (2010) Genome-wide survey and expression analysis suggest diverse roles of glutaredoxin gene family members during development and response to various stimuli in rice. DNA Res. 17: 353-367.

38. Raghuvanshi S, Kapoor M, Tyagi S, Kapoor S, Khurana P, Khurana JP, Tyagi AK (2010) Rice genomics moves ahead. Mol. Breed. 26: 257-273.

39. Sharma R, Kapoor M, Tyagi AK, Kapoor S (2010) Comparative transcript profiling of TCP family genes provide insight into gene functions and diversification in rice and Arabidopsis. J. Plant. Mol. Biol. Biotechnol. 1: 24-38.

40. Singh A, Giri J, Kapoor S, Tyagi AK, Pandey GK (2010) Protein phosphatase complement in rice: genome-wide identification and transcriptional analysis under abiotic stress conditions and reproductive development. BMC Genomics 11: 435.

41. Agarwal P, Kapoor S, Tyagi AK (2011) Transcription factors regulating the progression of monocot and dicot seed development. BioEssays 33: 189-202.

42. Chauhan H, Khurana N, Aggarwal P, Khurana P (2011) Heat shock factors in rice (Oryza sativa L.): Genome wide expression analysis during reproductive development and abiotic stress. Mol. Genet. Genomics 286 (2011) 171-187.

43. Giri J, Vij S, Dansana PK & Tyagi AK (2011) Rice A20/AN1 zinc-finger containing stress-associated proteins (SAP1/11) and a receptor-like cytoplasmic kinase (OsRLCK253) interact via A20 zinc-finger and confer abiotic stress tolerance in transgenic Arabidopsis plants. New Phytol. 191: 721-732.

44. Mathur S, Vyas S, Kapoor S, Tyagi AK (2011) The mediator complex in plants: structure, phylogeny and expression profiling of representative genes in a dicot (Arabidopsis thaliana) and a monocot (Oryza sativa) during reproduction and stress. Plant Physiol. 157: 1609-1627.

45. Ray S, Dansana PK, Giri J, Deveshwar P, Arora R, Agarwal P, Khurana JP, Kapoor S , Tyagi AK (2011) Modulation of transcription factor and metabolic pathway genes in response to water-deficit stress in rice. Funct. Integr. Genomics 11: 157-178.

46. Swapna L, Khurana R, Kumar SV, Tyagi AK, Rao KV (2011) Pollen-specific expression of Oryza sativa indica pollen allergen gene (OSIPA) promoter in rice and Arabidopsis transgenic systems. Mol. Biotechnol. 48: 49-59.

47. Garg R, Tyagi AK, Jain M (2012) Genome-wide analysis reveals overlapping and specific transcriptional responses to different plant hormones in rice. Plant Signal. Behav. 7: 951-956.

48. Khurana N, Chauhan H, Khurana P (2012) Cloning and expression analysis of a heat-inducible, myo-inositol-1-phosphate synthase (MIPS) gene from wheat and its comparative analysis in rice and Arabidopsis. Plant Cell Reports 31: 237-251.

49. Khurana R, Kapoor S, Tyagi AK (2012) Anthology of anther/pollen-specific promoters and transcription factors. Crit. Rev. Plant Sci. 31: 359-390.

50. Ray S, Kapoor S, Tyagi AK (2012) Analysis of transcriptional and upstream regulatory sequence activity of two environmental stress-inducible genes, NBS-Str1 and BLEC-Str8, of rice. Transgenic Res. 21: 351-366.

51. Sharma R, Agarwal P, Ray S, Deveshwar P, Sharma P, Sharma N, Nijhawan A, Jain M, Singh AK, Singh VP, Khurana JP, Tyagi AK, Kapoor S (2012) Expression dynamics of metabolic and regulatory components across stages of panicle and seed development in indica rice. Funct. Integr. Genomics 12: 229-248.

52. Giri J, Dansana PK, Kothari KS, Sharma G, Vij S, Tyagi AK (2013) SAPs as novel regulators of abiotic stress response in plants. BioEssays 35: 639-648.

53. Khurana R, Kapoor S, Tyagi AK (2013) Spatial and temporal activity of upstream regulatory regions of rice anther-specific genes in transgenic rice and Arabidopsis. Transgenic Res. 22: 31-46.

54. Khurana R, Kathuria H, Mukhopadhyay A, Kapoor S, Tyagi AK (2013) A 286 bp upstream regulatory region of a rice anther-specific gene, OSIPP3, confers pollen-specific expression in Arabidopsis. Biotech. Lett. 35: 455-462.

55. Nayar S, Sharma R, Tyagi AK, Kapoor S (2013) Functional delineation of OsMADS29 reveals its role in embryo and endosperm development by affecting hormone homeostasis. J Exp. Bot. 64: 4239-4253.

56. Singh A, Kanwar P, Pandey A, Tyagi AK, Sopory SK, Kapoor S, Pandey G (2013) Comprehensive genomic and expression profiling of phospholipase C gene family during abiotic stress and development in rice. PLoS One 8: e62494.

57. Dansana PK, Kothari KS, Vij S, Tyagi AK (2014) OsiSAP1 overexpression improves water-deficit stress tolerance in transgenic rice by affecting expression of endogenous stress related genes. Plant Cell Rep. 33: 1425-1440.

58. Gour P, Garg P, Jain R, Joshep S, Tyagi A, Raghuvanshi S (2014) Manually curated database of rice proteins. Nucl. Acids Res. 42: D1214-1221.

59. Sharma M, Singh A, Shankar A, Pandey A, Baranwal V, Kapoor S, Tyagi A, Pandey G (2014) Comprehensive expression analysis of rice Armadillo gene family reveals their functional diversity in abiotic stress and development. DNA Res. 21: 267-283.

60. Singh A, Kanwar P, Yadav A, Mishra M, Jha S, Baranwal V, Pandey A, Kapoor S, Tyagi A, Pandey G (2014) Genome-wide expressional and functional analysis of calcium transport elements during abiotic stress and development in rice. FEBS J. 281: 894-915.

61. Tyagi H, Jha S, Sharma M, Giri J, Tyagi AK (2014) Rice SAPs are responsive to multiple biotic stresses and overexpression of OsSAP1, an A20/AN1 zinc-finger protein, enhances the basal resistance against pathogen infection in tobacco. Plant Sci. 225: 68-76.

Future plans

A set of key genes and their functions revealed during our earlier studies on rice would become the nucleating points for the development of gene regulatory networks and initiate in-depth studies into landmark events during reproductive development. These landmark events include, (a) transition to flowering, (b) panicle branching, (c) determination of floral organs, (d) development of male and female reproductive organs, (e) meiosis, (f) pollen maturation, (g) embryo and endosperm development and (h) grain filling with starch. We aim to generate knowledge on molecular aspects of regulation of reproductive development in rice and develop precision tools for manipulation of biotechnologically relevant traits. Whenever the resource generated is validated, it will be deployed for trait enhancement.

The investigation will also be undertaken on forward molecular genetics of seed related traits. For this, selection and designing of large number of informative microsatellite and SNP markers from the target sequences and use these markers further in bi-parental linkage mapping and genetic association analysis would be undertaken. A correlation of the genotypic information (markers, novel genes/alleles and haplotypes) with differential transcriptome and epigenome profiling data and classical genetic inheritance studies to identify functionally relevant novel genes and alleles associated with seed size/weight in rice would be established. Efforts would be made to decipher the molecular and evolutionary mechanism of natural allelic/gene interactions influencing the seed size/weight variation in contrasting rice genotypes that have adapted to diverse agro-climatic conditions in India. The prime objective is not only to identify loci and functionally relevant molecular tags for seed size/weight through integrated epigenomics, transcriptomics and molecular genetics approaches, but also to utilize the seed size/weight trait-specific favourable natural allelic variants and superior novel gene combinations for marker-assisted genetic improvement of rice for seed size/weight and yield and eventually to produce high-yielding varieties.

Use of rice sequence for breeding in India

The available gold standard reference rice whole genome sequence has propelled the whole genome resequencing and transcriptome sequencing of diverse rice genotypes in recent years by use of next-generation sequencing (NGS) approaches in India. This in turn led to the development of enormous resources in the form of genomic (genic) simple sequence repeat (SSR) and single nucleotide polymorphism (SNP) markers at a genome-wide scale in indica rice. The large-scale validation and high-throughput genotyping of these identified informative genome/gene-based markers by employing various high-throughput array-based NGS and marker genotyping technologies in diverse natural Indian germplasm (core and mini-core) collections, advanced generation mapping and mutant populations of rice are underway. These efforts ensued many promising outcomes, including scanning of novel functional allelic variants from diverse rice genetic resources and understanding for molecular diversity, population genetic structure and domestication patterns, particularly among natural germplasm of rice. Consequently, these exertions assisted in construction of ultra-high density genetic linkage maps, QTL/eQTL mapping, fine mapping, candidate gene-based/genome-wide association study (GWAS) and map-based cloning for identifying potential genomic loci (major/minor QTLs, genes and alleles) associated with many qualitative and complex quantitative traits of agronomic importance in rice. Many successful endeavours have been made to introgress and pyramid the superior functional genes, major/minor QTLs and elite rare natural allelic variants regulating complex yield/quality component and stress tolerance traits (grain size and quality parameters, hybrid performance, bacterial leaf blight and blast resistance, and submergence and drought tolerance) into diverse rice genotypes (including Pusa Basmati1, Samba Mahsuri, Swarna, Pusa6A/6B, PRR78, Lalat, Tapaswani and IR64) using marker-assisted selection (MAS), multi-parent advanced generation intercross (MAGIC) and genomic selection for their genetic enhancement. This eventually led to development of certain diverse genetically-tailored high-yielding and climate resilient early maturing Indian rice varieties for sustaining food security.

Team of Indian Initiative for Rice Genome Sequencing.

From left to right: Drs. K. Gaikwad, T.R. Sharma, N.K. Singh, A. K. Tyagi, J.P. Khurana, T. Mohapatra, P. Khurana

Inaugural ceremony of 11th ISRFG hosted by India in 2013.

Left to right, Prof. J.P. Khurana, Prof. D. Singh, Prof. Q. Zhang, Dr. M.S. Swaminathan, Dr. M. Sharma, and Prof. A.K. Tyagi.