Transcriptome Assembly and SNP Identification in Pongamia pinnata (L.) Pierre using RNA-seq

Pongamia, or pongam, (Pongamia pinnata (L.) Pierre), is a familiar tree in many parts of India.  Outside of India, it is native to Southeast Asia and also occurs naturally in Australia.  Pongamia’s oil-rich seeds have sparked considerable attention as a source of high quality bio-oil.  We seek to better understand traits and biochemical pathways contributing to oil production and quality as well as adaptive vigor in varying environments.  Thus, we have begun to develop DNA markers for implementing marker accelerated breeding.  We conducted Next Generation (NextGen) sequencing using RNA-seq to simultaneously characterize expressed genes and identify candidate single nucleotide polymorphisms (SNPs).  RNAs were isolated from leaves of 72 seedlings and consolidated into eight pools of nine individuals.  From eight sequencing lanes on an Illumina HiSeq 2000 (100 bp paired-end reads), we generated ~40 Gbp of sequence.  After various high-stringency quality control steps, we processed 77.8M reads through the Trinity and Oases/Velvet assemblers, yielding ~57K and ~64K contigs of >300 bp.  The final assembly encompassed 56,691 unique contigs (“unigenes”) with an average length of 705 bp.  Sequence alignments with several reference genomes, including Glycine max, coupled with functional comparisons such as GO, revealed an expected assortment of expressed genes.  In addition, we identified ~98K candidate SNPs, with a frequency of about 1 in 400 bases.  As for other data-poor species, we conclude that RNA-Seq will be an effective tool for transcriptome assembly and SNP discovery to build infrastructure for marker accelerated breeding.