Preliminary SNP Discovery in Loblolly Pine (Pinus taeda L.) Exome

We are using the Agilent SureSelect Target Enrichment method to capture unigene-based targeted genomic sequences for SNP discovery. Sequencing libraries from the megagametophyte and embryo of a single 20-1010 loblolly pine seed were generated according to the Agilent SureSelect target enrichment protocol. 647,634 baits were designed using 35,550 unigenes provided by Dr. Chun Liang (Miami University, Oxford, Ohio) and used to hybridize the sequencing libraries. After capturing the targeted sequences, both barcoded sequencing libraries were pooled together and sequenced in Illumina HiSeq2000 using paired-end sequencing (2×100bp).

66,332,680 and 74,123,676 reads were obtained from the embryo and megagametophyte libraries, respectively. We performed de novo assembly with the filtered megagametophyte reads using CLC Genomics Workbench 5.5.1. 184,907 contigs (≈58Mb) were generated. The embryo reads were mapped to the megagametophyte contigs. Under relatively stringent condition, 30.61% of the embryo reads were mapped. 51,623 high quality SNPs were detected with a minimum number of reads equaled to 15 in a moving window, and a deviation from a 1:1 ratio of no more than 20%. The reads were also mapped to the unigenes. Under stringent mapping parameters, >92% of the unigenes were mapped. The unmapped unigenes were blasted against the 18Gb draft loblolly pine genome assembly. No significant hits were found for 168 unigenes.

We conclude that the rate of SNPs was about 0.0009 (which could be considered as a very conservative estimate). In addition, most unigenes were effective for baits design, but baits re-design is probably needed to improve their efficiency.