Open access
Announcement
12 September 2019

Draft Genome Sequence of Novel Metschnikowia sp. Strain JCM 33374, a Nectar Yeast Isolated from a Bumblebee

ABSTRACT

Here, we report the draft genome sequence of Metschnikowia sp. strain JCM 33374, a nectar yeast isolated from a bumblebee (Bombus diversus). The genome of 20.1 Mb is a naturally heterozygous diploid. Phylogenetic analysis with related taxa demonstrated that the strain is very likely a novel species.

ANNOUNCEMENT

The genus Metschnikowia (Saccharomycetaceae) consists of more than 80 validly described species of ascomycetous yeasts (1), characterized by the formation of needle-shaped ascospores as their sexual forms (2). Metschnikowia species show a high degree of ecological specialization (3), some of which are found at the plant-insect interface (47). We isolated Metschnikowia sp. strain JCM 33374 from nectar carried by a worker bumblebee (Bombus diversus) (Hymenoptera: Apidae) foraging flowers of red clover Trifolium pratense on a grassland in Sugadaira, Nagano Prefecture, Japan (36.523857°N, 138.348215°E). To retrieve nectar from a crop of the bumblebee, we gently squeezed her abdomen to regurgitate her crop content, which was then collected and stored using a sterile 10-μl microcapillary tube (Drummond Scientific, USA). We cultivated the strain in yeast-malt (YM) liquid medium (Difco, USA) at 20°C for 3 days, and the genomic DNA was extracted using the Genomic-tip 100/G extraction kit (Qiagen, USA). Genome sequencing was performed on the MinION system (Oxford Nanopore Technologies, UK) using an R9.4 flow cell with the rapid sequencing kit (SQK-RAD002) protocol, or on the MiSeq platform (Illumina, USA) using approximately 5% of a flow cell with the 2 × 300-bp paired-end protocol. The MinION system generated 105,151 reads (mean length, 5,514 bp), while the MiSeq platform generated 1,067,271 pairs of reads (mean length, 284 bp) after filtering by fastp v0.19.6 (8), both of which were processed for the following analyses. The combined assembly of the MinION and MiSeq reads was performed following the assembly-polish pipeline (https://github.com/nanoporetech/ont-assembly-polish), which integrated the assembler Canu v1.7 (9) for the long MinION reads with the consensus module Racon v1.3.1 (10) and the assembly improvement tool Pilon v1.22 (11) tailored for polishing based on the short Illumina reads.
The assembled draft genome consisted of 20,114,833 bp from 165 contigs, with a G+C content of 43.5%. The longest contig was 1,028,286 bp long, and the N50 value was 208,628 bp. Average coverages were 16.4× and 32.3× for the MinION and the MiSeq reads, respectively. This draft genome sequence contains 90.3% (1,473 complete and 72 fragmented) of the 1,711 benchmarking universal single-copy orthologs (BUSCOs) (BUSCO v3.0.2 [12]) using the OrthoDB v9 data set for Saccharomycetales. Protein-coding genes were annotated using AUGUSTUS v3.3.1 (13) with a Saccharomyces cerevisiae training set, which predicted 6,685 genes. tRNAscan-SE v2.0 (14) identified 240 tRNA genes. Genome Analysis Tool Kit (GATK v4.1.2.0) (15) best practices were performed to filter and accept a total of 148,417 single nucleotide polymorphisms (SNPs) and 30,795 insertion and deletions (indels). The observed distributions of allele depth ratios for all SNPs and indels exhibited a single peak at around 0.5, representing that the naturally heterozygous genome is diploid.
Phylogenetic analysis was performed to determine if the strain could be part of a novel species. A maximum likelihood tree was constructed using RAxML v8 (16) on the sequence of the D1/D2 domain of the large-subunit (LSU) rRNA gene from the draft genome with those from the Metschnikowia clade available in the DDBJ/ENA/GenBank database. The phylogenetic tree revealed that the strain located in the Metschnikowia clade but was different from any described Metschnikowia species. The sequence of the LSU D1/D2 from the strain was validated by Sanger sequencing and showed 6.3% divergence from that of the closely related species Metschnikowia lachancei (17). For ascomycetous yeasts, more than 1% divergence in LSU D1/D2 represents the threshold used to discriminate a species (18). Therefore, the strain is very likely a novel species.

Data availability.

The draft genome sequence and annotation data of Metschnikowia sp. strain JCM 33374 have been deposited at DDBJ/ENA/GenBank under the accession number BIMT00000000. The version described in this paper is the first version, BIMT01000000. The raw reads were deposited in the SRA/DRA/ERA under the accession numbers DRA008301 and DRA008302. The strain is available from the Japan Collection of Microorganisms, RIKEN BioResource Research Center (Tsukuba, Ibaraki, Japan), under strain number JCM 33374.

ACKNOWLEDGMENTS

This work was supported by the Japan Society for the Promotion of Science KAKENHI (grant 15K12256) to A.S.H.
Computations were partially performed on the NIG supercomputer at the ROIS National Institute of Genetics. We thank the students of the filed courses 2016 at Sugadaira Research Center, University of Tsukuba, for their assistance in collecting samples. We also thank Muneki Yamada for his support.

REFERENCES

1.
Lachance MA. 2016. Metschnikowia: half tetrads, a regicide and the fountain of youth. Yeast 33:563–574.
2.
Lachance MA. 2011. Metschnikowia Kamienski (1899), p 575–620. In Kurtzman CP, Fell J, Boekhout T (ed), The yeasts, a taxonomic study, 5th ed, vol 2. Elsevier Science, London, United Kingdom.
3.
Guzmán B, Lachance MA, Herrera CM. 2013. Phylogenetic analysis of the angiosperm-floricolous insect-yeast association: have yeast and angiosperm lineages co-diversified? Mol Phylogenet Evol 68:161–175.
4.
Giménez-Jurado G. 1992. Metschnikowia gruessii sp. nov., the teleomorph of Nectaromyces reukaufii but not of Candida reukaufii. Syst Appl Microbiol 15:432–438.
5.
Brysch-Herzberg M. 2004. Ecology of yeasts in plant-bumblebee mutualism in central Europe. FEMS Microbiol Ecol 50:87–100.
6.
de Vega C, Guzman B, Lachance MA, Steenhuisen SL, Johnson SD, Herrera CM. 2012. Metschnikowia proteae sp. nov., a nectarivorous insect-associated yeast species from Africa. Int J Syst Evol Microbiol 62:2538–2545.
7.
Herrera CM, Pozo MI, Medrano M. 2013. Yeasts in nectar of an early-blooming herb: sought by bumble bees, detrimental to plant fecundity. Ecology 94:273–279.
8.
Chen S, Zhou Y, Chen Y, Gu J. 2018. fastp: an ultra-fast all-in-one FASTQ preprocessor. Bioinformatics 34:i884–i890.
9.
Koren S, Walenz BP, Berlin K, Miller JR, Bergman NH, Phillippy AM. 2017. Canu: scalable and accurate long-read assembly via adaptive k-mer weighting and repeat separation. Genome Res 27:722–736.
10.
Vaser R, Sovic I, Nagarajan N, Sikic M. 2017. Fast and accurate de novo genome assembly from long uncorrected reads. Genome Res 27:737–746.
11.
Walker BJ, Abeel T, Shea T, Priest M, Abouelliel A, Sakthikumar S, Cuomo CA, Zeng Q, Wortman J, Young SK, Earl AM. 2014. Pilon: an integrated tool for comprehensive microbial variant detection and genome assembly improvement. PLoS One 9:e112963.
12.
Simão FA, Waterhouse RM, Ioannidis P, Kriventseva EV, Zdobnov EM. 2015. BUSCO: assessing genome assembly and annotation completeness with single-copy orthologs. Bioinformatics 31:3210–3212.
13.
Stanke M, Steinkamp R, Waack S, Morgenstern B. 2004. AUGUSTUS: a Web server for gene finding in eukaryotes. Nucleic Acids Res 32:W309–W312.
14.
Lowe TM, Eddy SR. 1997. tRNAscan-SE: a program for improved detection of transfer RNA genes in genomic sequence. Nucleic Acids Res 25:955.
15.
McKenna A, Hanna M, Banks E, Sivachenko A, Cibulskis K, Kernytsky A, Garimella K, Altshuler D, Gabriel S, Daly M, DePristo MA. 2010. The Genome Analysis Toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data. Genome Res 20:1297–1303.
16.
Stamatakis A. 2014. RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics 30:1312–1313.
17.
Giménez-Jurado G, Kurtzman CP, Starmer WT, Spencer-Martins I. 2003. Metschnikowia vanudenii sp. nov. and Metschnikowia lachancei sp. nov., from flowers and associated insects in North America. Int J Syst Evol Microbiol 53:1665–1670.
18.
Kurtzman CP, Robnett CJ. 1998. Identification and phylogeny of ascomycetous yeasts from analysis of nuclear large subunit (26S) ribosomal DNA partial sequences. Antonie Van Leeuwenhoek 73:331–371.

Information & Contributors

Information

Published In

cover image Microbiology Resource Announcements
Microbiology Resource Announcements
Volume 8Number 3712 September 2019
eLocator: 10.1128/mra.00704-19
Editor: Jason E. Stajich, University of California, Riverside

History

Received: 21 June 2019
Accepted: 23 August 2019
Published online: 12 September 2019

Contributors

Authors

Sugadaira Research Station, Mountain Science Center, University of Tsukuba, Ueda, Nagano, Japan
Faculty of Symbiotic Systems Science, Fukushima University, Fukushima, Fukushima, Japan
Ryosuke Imai
Sugadaira Research Station, Mountain Science Center, University of Tsukuba, Ueda, Nagano, Japan
Iriomote Station, Tropical Biosphere Research Center, University of the Ryukyus, Nishihara, Okinawa, Japan
Rikiya Endoh
Microbe Division, Japan Collection of Microorganisms, RIKEN BioResource Research Center, Tsukuba, Ibaraki, Japan
Moriya Ohkuma
Microbe Division, Japan Collection of Microorganisms, RIKEN BioResource Research Center, Tsukuba, Ibaraki, Japan
Sugadaira Research Station, Mountain Science Center, University of Tsukuba, Ueda, Nagano, Japan

Editor

Jason E. Stajich
Editor
University of California, Riverside

Notes

Address correspondence to Akira S. Hirao, [email protected].

Metrics & Citations

Metrics

Note:

  • For recently published articles, the TOTAL download count will appear as zero until a new month starts.
  • There is a 3- to 4-day delay in article usage, so article usage will not appear immediately after publication.
  • Citation counts come from the Crossref Cited by service.

Citations

If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. For an editable text file, please select Medlars format which will download as a .txt file. Simply select your manager software from the list below and click Download.

View Options

Figures and Media

Figures

Media

Tables

Share

Share

Share the article link

Share with email

Email a colleague

Share on social media

American Society for Microbiology ("ASM") is committed to maintaining your confidence and trust with respect to the information we collect from you on websites owned and operated by ASM ("ASM Web Sites") and other sources. This Privacy Policy sets forth the information we collect about you, how we use this information and the choices you have about how we use such information.
FIND OUT MORE about the privacy policy