Complete Genome Sequences of Four Isolates of Plutella xylostella Granulovirus.

Granuloviruses are widespread pathogens of Plutella xylostella L. (diamondback moth) and potential biopesticides for control of this global insect pest. We report the complete genomes of four Plutella xylostella granulovirus isolates from China, Malaysia, and Taiwan exhibiting pairs of noncoding, homologous repeat regions with significant sequence variation but equivalent length.

GENOME ANNOUNCEMENT

Plutella xylostella L. (diamondback moth) is a globally important insect pest of Brassica crops that is widely resistant to insecticides incurring control costs up to five billion U.S. dollars annually (1–3). Plutella xylostella granuloviruses (PlxyGV) have potential use as biopesticides to manage insecticide resistance and improve pest management (2, 4, 5). Here, we present complete genome sequences of four isolates of PlxyGV from China, Malaysia, and two from Taiwan (6, 7).

Isolates were passaged through P. xylostella larvae and occlusion bodies purified by centrifugation, followed by incubations in 1 M sodium carbonate (Na2CO3) at room temperature for 5 min and 1% N-lauryl sarcosine at 37°C for 15 min. DNA was then purified using an Isolate II Genomic DNA kit (catalogue no. BIO-52067, Bioline) from step 4 according to manufacturer’s instructions.

Genomic DNA was prepared for sequencing using the Nextera XT kit and medium-output flow cell on an Illumina Next-Seq 500 with 150 bp paired-end sequencing. Raw sequence data comprised 12,852,411 reads (PlxyGV-C, China); 8,878,618 reads (PlxyGV-T, Taiwan); 12,251,888 reads (PlxyGV-K, Taiwan); and 16,077,717 reads (PlxyGV-M, Malaysia), representing average genome coverage of 19,088×, 13,186×, 18,196×, and 23,878×, respectively.

Read inspection, trimming, and genome assembly used the method of Noune and Hauxwell (8). Reads were analyzed and trimmed using FastQC version 0.11.3 (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/) and FastX trimmer version 0.0.13 (http://hannonlab.cshl.edu/fastx_toolkit). Genomes were assembled de novo using Tadpole (BBMap 35.49) (http://sourceforge.net/projects/bbmap) with kmer values generated by Kmergenie (9) and mapping to the NC_002593.1 reference (10) using BWA version 0.7.12 (11) and SAMtools version 1.2 (12). The de novo assembled contigs, BWA generated mapping, and trimmed reads were merged into a single FastQ file and then re-mapped to the NC_002593.1 reference using the Geneious R9 mapper (13) with low to medium sensitivity and 5 iterations. Gaps were filled manually from Sanger sequences. All four genomes showed a high degree of similarity, with 40.7% G+C content and sequence homology of 99.9%. Open reading frames (ORFs) were predicted using the Geneious R9 live annotation tool and compared to the NC_002593.1 reference with the larger of overlapping ORFs selected. One hundred eighteen ORFs were predicted for all four PlxyGV isolates, two fewer than the NC_002593.1 reference genome. The completed genomes are 100,980 bp (PlxyGV-C), 100,978 bp (PlxyGV-T), 101,004 bp (PlxyGV-K), and 100,980 bp (PlxyGV-M) in length.

Genomic differences arise in the position of ORF73 (which shares 58.6% nucleotide sequence identity with AcMNPV ORF91) and is truncated in PlxyGV-K. The regions of greatest sequence variation occur within two pairs of noncoding regions, which are almost identical in length (pair one, 2,596 bp and 2,516 bp; pair two, 1,340 bp and 1,414 bp) in the four isolates and NC_002593.1 reference genome. These are homologous repeat regions that are common features within baculovirus genomes (14).

Nucleotide sequence accession numbers.

This whole-genome shotgun project has been deposited in DDBJ/EMBL/GenBank under the accession numbers KU529791 (PlxyGV-C), KU529792 (PlxyGV-K), KU529793 (PlxyGV-M), and KU529794 (PlxyGV-T). The versions described in this paper are the first versions, KU529791.1, KU529792.1, KU529793.1, and KU529794.1.