Draft Genome Sequence of “Candidatus Sulcia muelleri” Strain KPTW1 from Kolla paulula, a Vector of Xylella fastidiosa Causing Pierce’s Disease of Grapevine in Taiwan

In 2002, Pierce’s disease (PD) of grapevine caused by Xylella fastidiosa was found in several municipalities in Taiwan (1, 2). Kolla paulula was confirmed to be an insect vector of X. fastidiosa (3, 4). The insect occurs in all vineyards and is distributed over the medium- and low-altitude areas in Taiwan (3, 5). PD is endemic in California and the southeastern United States and is reportedly transmitted by xylem-feeding leafhoppers, such as blue-green sharpshooter (BGSS; Graphocephala atropunctata) and glassy winged sharpshooter (GWSS; Homalodisca vitripennis). Studies on BGSS and GWSS have revealed the presence of several endosymbionts, including “Candidatus Sulcia muelleri,” which is important for insect growth and development (6 – 8). In this study, we report the whole-genome sequence of “Ca. Sulcia muelleri” in K. paulula from Taiwan.

A colony of K. paulula (female/male) was established and maintained on Commelina diffusa under laboratory conditions (25°C, 60% RH, 13:11 light/day [L:D]) at the Taiwan Agricultural Research Institute in 2016. DNA was extracted from a single insect using a DNeasy blood and tissue kit (Qiagen, Valencia, CA). Whole-genomic DNA was amplified using an illustra GenomiPhi version 2 DNA amplification kit (GE Healthcare, Waukesha, WI) and sequenced using MiSeq (2 × 150) format (Illumina, San Diego, CA) after preparation using the Illumina TruSeq DNA PCR-free library prep kit.

A total of 28,327,432 reads (151 bp each) with average Q value above 30 were generated. De novo assembly using default settings (word size = 20, bubble size = 50) was performed with CLC Genomics Workbench. The top 20 largest contigs ranging from 251,844 to 12,494 bp were collected. The top 200 bp at the 5′ end of each contig were used for BLASTn search against GenBank nr database (version 225.0) containing 21 whole-genome sequences of “Ca. Sulcia muelleri.” Three contigs hit the whole-genome sequence of “Ca. Sulcia muelleri” from BGSS (GenBank accession number CP008986) (6), with 100% query coverage and 93% identity. CP008986 was used for reference mapping (length fraction = 100, similarity fraction = 90) to assemble the “Ca. Sulcia muelleri” genome sequence from K. paulula using the MiSeq data with CLC Genomics Workbench. Gap closures among reference mapping contigs were first made based on information in de novo contigs, followed by read walking/extension. Briefly, 100 Ns were added at either end of a targeted contig. The contig was used for reference mapping with MiSeq reads (length fraction = 50, identity fraction = 100). Overlapping reads (50 bp minimum) were identified and added for contig extension. The extended contig was used to search for overlapping with other long contigs for extension if found. The read walking procedure continued until no overlapped reads were found.

The draft genome of “Ca. Sulcia muelleri,” designated strain KPTW1, consists of 9 contigs from 1,695 to 137,380 bp, with a total size of 253,942 bp (average nucleotide coverage, 64×) and a GC content of 22.7%. Sequence annotation was performed using the RAST server (http://rast.nmpdr.org/) (9). The draft genome was predicted to have 237 protein-coding genes and 34 RNA genes. The KPTW1 genome is 96.4% of that of the reference BGSS (244,618 bp).

This research was supported by TARI (grant 107AS-8.4.1-CI-C1) and a USDA-ARS base fund. The mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture.