Targeted Transcriptome Analysis Using PacBio Sequencing to Dissect Multi-Gene Families Encoding NBS-LRR Resistance Proteins in Lettuce

The majority of disease resistance (R) genes cloned to date encode nucleotide-binding site, leucine-rich repeat (NBS-LRR) proteins.  These genes comprise one of the largest gene families in plants, are ancient in origin, and found in multiple copies that are often clustered in the genome. They are large genes ranging from 2 to 15 kb with transcripts that range from ~1.5 to 6 kb and are lowly expressed.  Some genes encode subsets of domains and/or additional domains.  Alternative splicing can also occur.  Obtaining full-length sequences of specific genes is informative for studying the evolution and mechanisms of disease resistance as well as for breeding applications. However, the dissection of multi-gene families with high sequence similarity is challenging or impossible with short-read sequencing data alone. We utilized PacBio sequencing to study the genetic architecture and diversity of R genes in lettuce and the effect of RNAi constructs targeting specific NBS-LRR encoding gene families. Size-selected cDNA libraries generated from replicates of six different genotypes and six RNAi transgenic lines with different NBS-LRR gene families targeted for silencing were sequenced using 28 SMRT cells, yielding an average of ~100.2 K transcript sequences per SMRT cell. Transcript sequences up to 10 kb were obtained. On average 1% of the transcripts were identified as NBS-LRR encoding genes. This transcriptome data facilitated comparative expression analyses of NBS-LRR encoding genes in the both RNAi and wild-type genotypes. The data was also informative for refining the annotation of the lettuce genome.