1. Abstract To understand the population dynamics in rice we conducted detailed sequence study in 32 accessions of rice (10 japonica, 7 indica, 10 Asiatic and five other wild species). We sequenced and aligned ~1 kb region representing 20 ESTs distributed on chromosomes 1, 2, 3 and 4. Analysis of approximately 18.3 kb (5.5 kb coding and 12.8 kb noncoding) sequence revealed very low level of polymorphism among japonica cultivars (  =4  10 -4 ) followed by indica (  =20.2  10 -4 ) as against rufipogon accessions (  = 47.8  10 -4 ). Distance between japonica and indica (D IJ = 11  10 -4 ) was low as compared to African accessions to indica (D IA =31.4  10 -4 ) and African accessions to japonica (D JA =25.6  10 -4 ). Ratio of  in Asiatic accessions to distance between Asian and African groups (D AA ) crept reducing from telomeric to centromeric regions, suggesting reduction in polymorphism in terms of evolution towards centromere, possibly as a result of selection sweep. Multiple sequence alignment has revealed number of informative SNPs segregating within/between indica, japonica and wild species. Bulk eco-TILLING has been applied for faster polymorphism detection in rice. Employing eco-TILLING across 48 accessions revealed LD decay after ~70 kbp.

2. Objectives  Investigation on extent of polymorphism at sequence level in rice  Detection of useful SNPs to use them in rice breeding programme Materials  Rice is one of the most important food crop across the world  Rice has two main groups: ssp. indica & ssp. japonica besides O. glaberrima (African spp.)  O. rufipogon is considered as wild progenitor of domesticated rice  Rice is only cereal crop which has complete genome sequence from both ssp. Indica and japonica  Evolutionary forces that shaped genomic diversity is little known  We have little information on SNP polymorphism, linkage disequilibrium and other population genomic information in rice Introduction

3. Methodology  Sequencing & alignment-polymorphism detection  Eco-TILLING-polymorphism detection-sequencing & alignment-polymorphism confirmation

4. Phylogenetic relationship among O. rufipogon accessions based on Eco-TILLING Phylogenetic relationship among the accessions based on sequence information Japonica & rufipogon Indica & rufipogon African rufipogon Ind.Jap.Cult.AsiaRuf.Afr. Ind.-11--2.331.4 Jap.---8.125.6 Cult.--0.424 Asia--13.3 Ruf.-15.3 Genetic distances between various groups of Oryza Values in the table are  10 -4 Results African accessions are distinct out group in Oryza spp.

5. Summary of DNA polymorphism in rice   Values (  10 -4 ) Total Indica Japonica Cultivated Asiatic rufipogon African Total Indica Japonica Cultivated Asiatic rufipogon African Total Indica Japonica Cultivated Asiatic rufipogon African 0 1 2 3  Asia /D AA Chr. 1Chr. 3 Chr. 4 D AA : distance between Asian and African groups Entire sequenceCoding regionNoncoding region  Japonica varieties have a very narrow genetic base  O. Rufipogon retains huge untapped polymorphism Has selection sweep led to reduction in polymorphism to distance ratio toward centromere?

6. Indica G A C G A T A G T C T - A C G G Japonica A G T C G A G T A - C * T T A G African G A T G A T A T A C T - A C G G Indica Ile Thr Thr Thr Japonica Asp Thr Thr Gly *AACAGAATTCC 125401127116127374127611126823127724 AK121680 exon 125556 127218 AC119747 Exon UTR AK071921 AK060370 Phosphoribosylanthranilate transferase ??? Does mutation hotspot at AU162368 has any role in subspeciation of Oryza??  Eco-TILLING also confirmed this unusual dense distribution of mutation  Value of  is 93.7  10 -4  Distances calculated on the basis of sequence information for the locus abnormally exceeds the general trend Japonica Indica D IJ D IR D JR D IAf D JAf 137.213.570.733.5127 Values are in  10 -4

7. Distribution of some informative SNPs across genotypes Application of SNPs in germplasm characterization

8. Application of eco-TILLING in SNP detection

9. Linkage disequilibrium decay estimation using eco-TILLING Conduct Eco-TILLING of diverse genotypes Detect and position polymorphism and measure LD decay Loci 12 3 4567 Indica Japonica Rufipogon 700 channel 800 channel Eco-TILLING of japonica, indica and rufipogon accessions with reference to Nipponbore Indica Japonica Rufipogon LD decay occurs after 70 Kbp in rice LD mapping can be efficiently done in rice with identification of informative SNPs by combining bulk eco-TILLING and sequencing More efforst will have to be made for detection of informative SNPs in japonica accessions AP003260 33.7 Mb (~100 kb region) R2R2 Conclusion

10. Acknowledgement Steven Hanikoff, University of Washington, Seattle Bradley Til, University of Washington, Seattle Luca Comai, University of Washington, Seattle For Post-doc position on DNA polymorphism in rice contact R. Terauchi at terauchi@mail.ibrc.or.jp