RICE GENOME PROJECT
(Indian Initiative For Rice Genome Sequencing)
National Research on Plant Biotechnology, IARI
New Delhi-110012, INDIA
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Integral Physical Map of INDIAN REGION

BAC based Minimum Tilling Path of IARI 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.

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.

 

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.

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 RGP.

The detailed Physical Maps are shown below :

1. Integral Physical Map of INDIAN REGION

2. BAC based Minimum Tilling Path of IARI REGION

  
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