Rice Genetics IV - IRRI books - International Rice Research Institute

the development of a fingerprint DB. Eighty-six percent of the clones were successfullyfingerprinted and assembled into 1,039 contigs (FPC parameters: cutoff = 10 –12 ,tolerance = 7) and 2,924 singletons. An average contig contained 59 BAC clones ofabout 380 kb in size.Genome-anchoring databaseTo anchor the 1,039 contigs to the rice genetic map, we used three approaches. Thefirst was to develop probes from mapped restriction fragment length polymorphism(RFLP) markers and hybridize these markers to high-density hybridization filters representingboth BAC libraries. Second, overgo primers (http://genome.wustl.edu/gsc/overgo/overgo.html) were developed from STC (as described below) anchored contigs(step 1) to extend the contigs. Third, the STCs were used to tentatively anchor contigsbased on sequence homology with sequenced RFLP markers—in silico anchoring(Yuan et al 2000). Using these approaches, we have tentatively anchored 472 contigsto the rice genetic map containing 215–234 Mb of DNA or more than 50% of the ricegenome.The complete integrated fingerprint and anchoring databases are publicly availableusing WebFPC (Soderlund et al, unpublished) at www.genome.clemson.edu.Sequence-tagged connector database (STC-DB)To develop STC-DB, we attempted to sequence both ends of every rice DNA insert inthe BAC libraries described above. DNA was isolated from 63,729 BAC clones andend-sequenced using a high-throughput DNA sequencing protocol (Yu and Wing,unpublished). Table 2 summarizes the results. An average of 87% of all BAC endswere successfully sequenced, resulting in a total of 41 Mb of high-quality sequence(phred 20 or greater) being released to the dbGSS (database of Genome Survey Sequence)section of Genbank. On average, our results generated about 1 STC every 3.9kb across the rice genome.To obtain an early glimpse of the rice genome and to provide rice breeders with apotentially new set of genetic markers, we mined the HindIII STC data set for transposableelement and simple sequence repeat content.Figure 2 summarizes our analysis of the transposable element content of the riceHindIII STC-DB. Approximately 13% of the STCs have sequences similar to class I,Table 2. Publicly available rice sequence-tagged connectors (http://www.genome.clemson.edu). Nipponbare HindIII and EcoRI bacterial artificial chromosome (BAC)libraries, 40.67 Mb of high-quality a rice genomic sequence, 9.5% of the total ricegenome, 3.9 kb STC –1 .BAC Total rxn Successful (%) Av HQ a (bp)OSJNBa b 73,362 63,432 (86.5) 387OSJNBb b 54,097 47,006 (86.9) 343Total 127,459 110,438 (86.6) 368aNo. of bases having phred value ≥20. b OSJNBa is HindIII and OSJNBb is EcoRI BACclones.218 Wing et al