Progress in Sequencing the Genome of an Invasive Polyploid Weed (Leafy Spurge)

Leafy spurge is a perennial invasive weed that primarily infests range and recreation land in the great plains of the US and Canada. It has a chromosome number of 60 and is thought to be an auto-allo hexaploid. Flow cytometry indicated a haploid genome size of 2.1 Gb. Numerous microarrays and recent RNAseq experiments have been done on leafy spurge to identify genes responsive to various developmental programs and environmental conditions including drought stress, shifts from native to invasive environments and most prominently changes associated with shifts through seasonal bud dormancy states. Many clusters of coordinately regulated genes have been identified in these experiments and it would be desirable to identify the transcription factors involved in regulating these conditionally-responsive genes. Identifying common cis acting elements or performing phylogenetic foot-printing could identify transcription factors involved in these processes. However until recently few promoter sequences were available. We have used Illumina technology to sequence the leafy spurge genome to ~30X combined coverage from 4 small insert libraries (ranging from 270-400 bases). We used iPlant resources in attempts to assemble these sequences. SOAP-de novo was used in the Discovery Environment (DE) yielding only small contigs and scaffolds. However, BLAT resources in the DE identified Illumina sequences conserved between leafy spurge and three other related and fully sequenced species (poplar, cassava, and castor bean) as well as those sequences that were similar to the 19,000+ leafy spurge unigene ESTs from a Sanger-sequenced multi-tissue/environment library. Trinity was run in the DE on these conserved sequences and yielded 170K contigs with an N50 of over 600 bases. The promoter and intron sequences of select genes were filled in using the program PRICE-TI developed by Dr. Graham Ruby in the DeRisi lab (UCSF/HHMI) using Atmosphere resources. Promoter analyses of these select genes identify several interesting and conserved sequences potentially involved in crosstalk between environmental stress and circadian regulation in leafy spurge.