India has contributed to the International Rice Genome Sequencing Project by sequencing chromosome 11 through scientists at the University of Delhi and chromosome 12 through scientists at the Indian Agricultural Research Institute. Indian scientists developed a physical map of the target regions, screened a BAC library to identify relevant BAC clones, constructed BAC contigs to cover 70% of the minimum tiling path, and worked to fill gaps in the physical map through chromosome walking and identifying additional BACs. Purity of identified BACs was also checked to ensure high quality sequencing.

1. India’s
contribution in Rice
Genome Sequencing

2. HOW IT STARTS
 Rice (Oryza sativa) is one of the most important crops in the world.
Rice, wheat, and maize together account for about half of the world's
food production, and rice itself is the principal food of half of the
world's population
 The International Rice Genome Sequencing Project (IRGSP) began in
September 1997, at a workshop held in conjunction with the
International Symposium on Plant Molecular Biology in Singapore.
Scientists from many nations attended the workshop and agreed to an
international collaboration to sequence the rice genome.
 The IRGSP sequenced the genome of a single inbred cultivar, Oryza
sativa ssp. japonica cv. Nipponbare, and adopted a hierarchical clone-
by-clone method using bacterial and P1 artificial chromosome clones
(BACs and PACs, respectively).
 India, previously an unfunded member of the IRGSP, has a new Rice
Genome Program (represented by Akhilesh Tyagi of the University of
Delhi and Nagendra Singh of the Indian Agricultural Research
Institute) and will begin work on chromosome 11.

3. PERSONS FROM INDIA
 Indian Initiative for Rice Genome Sequencing (IIRGS),
University of Delhi South Campus (UDSC) for
chromosome no. 11
 S. Raghuvanshi, A. Mohanty, A. K. Bharti, A. Gaur11, V.
Gupta, D. Kumar, V. Ravi, S. Vij, A. Kapur, Parul Khurana,
Paramjit Khurana, J. P. Khurana, A. K. Tyagi, (Principal
Investigator);
 Indian Initiative for Rice Genome Sequencing (IIRGS),
Indian Agricultural Research Institute (IARI) for
chromosome no. 12
 K. Gaikwad, A. Singh, V. Dalal, S. Srivastava, A. Dixit, A. K.
Pal, I. A. Ghazi, M. Yadav, A. Pandit, A. Bhargava, K.
Sureshbabu, K. Batra, T. R. Sharma, T. Mohapatra, N. K.
Singh, (Principal Investigator);

4. 1. Integral Physical Map of INDIAN REGION
 Two scientists from Delhi University obtained the first set of physical mapping
data for our region while getting their three-months training at Tsukuba, Japan.
Dr. T. Mohapatra from the IARI further enriched this map using 13 new
markers from the Cornell and RGP maps and has filled many of the gaps
during his training at Clemson, USA in Oct.- Dec 2000.
 In order to find BAC clones belonging to the Indian region from the CUGI BAC
libraries, the first task was to design suitable probes to identify the DNA
clones required for the Indian programme.
 This involved downloading of DNA sequences of about 250 RFLP markers and
BAC ends already available in different web-sites.
 Blast search was carried out using each of these sequences to find out the
extent of redundancy.
 Relatively non-redundant sequences were selected and used to design a pair of
overlapping oligos (overgos) for each sequence.
 For this, a computer package "overgo maker" was used.
 A total of 60 pairs of overlapping oligos were synthesized. By Klenow filling, 60
(-32P labeled 40 nucleotide long overgo probes were designed and used to
screen BAC library

5. 2. BAC based Minimum Tilling Path
of IARI REGION
 BAC library consisted of 36,864 clones (about 132 kb each) of rice genomic DNA.
 These clones were spotted onto nylon membranes using a robot (Q-BOT) and allowed to
grow into micro-colonies overnight.
 The membranes were processed and used for pre-hybridization at 60 o C overnight.
 Each of the 60 overgo probes were hybridized with these membranes separately at 60 o C
overnight.
 The membranes were then washed and exposed to phosphor screens, which were
scanned using a Phosphor imager (Molecular Dynamics).
 The results of the hybridization were obtained and stored using Image Quant computer
package.
 The BAC DNA was isolated following a modified alkaline lysis method and digested
overnight using Hind III restriction enzyme.
 The restriction fragments were separated on a 1% agarose gel for 16 h at 60 volt with
provision for buffer circulation.
 The gels were stained using syber gold stain. The scanning of gels were done using the
computer package Image using Fluor-S imaging system (Bio-rad).
 Based on the lane size standard, the molecular weight of each restriction fragments was
estimated for construction of BAC contigs.

6.  The addresses of the BAC clones hit by the overgo probes were called and a data file was
created in the Microsoft Excel.
 This data file was then imported to a computer package called fingerprinted contigs (FPC)
for finding the BAC contigs belonging to the chromosomal region of interest for India.
 Based on the hybridization of 60 overgo probes with 120 BAC membranes, 12 BAC contigs
could be placed against the genetic position of the overgo probes.
 A total of 117 BAC clones spread over the chromosomal region assigned to India were
identified which constitute about 70 % of the minimum tiling path.
 The physical map constructed for the chromosomal region had several gaps. An effort was
made to fill the gaps largely by chromosome walking from the BAC end sequences.
 By repeated screening of BAC library using overgo probes derived from BAC end sequences.
 Three of the gaps could be closed. One of these was major and contained disease resistance
analogue sequence.
 Another strategy was also designed for gap filling employing yeast artificial chromosome
(YACs). For this YACs were isolated from Yeast and the target regions corresponding to the
gaps could be amplified by asymmetric PCR.
 These sequences can be used as probes to identify BAC contigs located in the region of gaps
in the current physical map.

7.  Purity of each of the PACs and BACs belonging to the
region assigned to the IARI has been checked by
fingerprint analysis of at least 8 colonies from each
BAC. Some of the BACs were found to be mixtures and
these have been either discarded and new supply is
obtained from Clemson and RGP2