Two New Indica Platinum Standard Reference Genomes

Rice, as a human food, including subspecies japonica and indica, is one of the most important crop plants in the world since it provides more than 20% of the caloric intake of half of the world’s population. Massive amounts of sequence data for rice have been generated by the research community over the past two decades. However, only one high-quality reference genome sequence (RefSeq) is  available (for the japonica subspecies) which was produced using Sanger chemistry and a clone-by-clone strategy. The rice community is eager to have several high-quality reference genomes, especially from the subspecies indica for which none currently exists. Immediately, an indica RefSeq could be used as anchor genetic traits that would point to the determination of genes required for the high performance of the crop.  Recently, we integrated a set of methods using the most advanced technologies currently available to produce what we are calling “Platinum Standard Reference Sequences” (PSRefSeq). This integration (combining large-insert BAC libraries, Whole Genome Profiling™ Physical Maps, PacBio sequencing of pooled BACs and other useful resources) was used to generate 2 PSRefSeqs from two important Chinese indica varieties – Zhenshan 97 and Minghui 63. In addition, we established an integrated informatics platform to manage all data generated during the production and assembly phase of our PSRefSeq system. For this project, we selected a minimum tiling path of ~4000 BACs from each variety, mixed 12 to 32 random clones per pool, and sequenced each pool on one or two SMRTcells. After the initial production sequencing over a 9-month period, >90% of these BACs were fully circularized, achieving a staus that are considered finished (phase III) BAC sequences. Only a few repeated experiments were required for those failed (uncircularized) BACs. With the fully sequenced BACs, we were able to assemble Zhenshan 97 and Minghui 63 sequences into 340 and 216 gap-free contigs covering ~337 Mb and ~357 Mb of their genomes (N50 length: ~1.6 Mb and ~2.9 Mb), respectively. From the ongoing project, we are confident that the final assembly results will yield two reference genomes of high quality, large contigs and error-free sequences. Updated methodology and data will be discussed.