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High-Quality Genome Assembly and Annotation Resource of Botryosphaeria dothidea Strain BDLA16-7, Causing Trunk Canker Disease on Chinese Hickory

2021; American Phytopathological Society; Volume: 106; Issue: 3 Linguagem: Inglês

10.1094/pdis-08-21-1623-a

1943-7692

Jiandong Bao, Qianqian Wu, Jianqin Huang, Chuanqing Zhang,

Plant-Microbe Interactions and Immunity

HomePlant DiseaseVol. 106, No. 3High-Quality Genome Assembly and Annotation Resource of Botryosphaeria dothidea Strain BDLA16-7, Causing Trunk Canker Disease on Chinese Hickory PreviousNext RESOURCE ANNOUNCEMENT OPENOpen Access licenseHigh-Quality Genome Assembly and Annotation Resource of Botryosphaeria dothidea Strain BDLA16-7, Causing Trunk Canker Disease on Chinese HickoryJiandong Bao, Qianqian Wu, Jianqin Huang, and Chuan-Qing ZhangJiandong Baohttps://orcid.org/0000-0003-3423-5118Department of Plant Pathology, Zhejiang Agriculture and Forestry University, Hangzhou 311300, ChinaFujian University Key Laboratory for Plant-Microbe Interaction, College of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou, 350002, China, Qianqian WuDepartment of Plant Pathology, Zhejiang Agriculture and Forestry University, Hangzhou 311300, China, Jianqin HuangState Key Laboratory of Subtropical Silviculture, Zhejiang Agriculture and Forestry University, Hangzhou, 311300, China, and Chuan-Qing Zhang†Corresponding author: C. Q. Zhang; E-mail Address: cqzhang@zafu.edu.cnhttps://orcid.org/0000-0002-0493-1959Department of Plant Pathology, Zhejiang Agriculture and Forestry University, Hangzhou 311300, China AffiliationsAuthors and Affiliations Jiandong Bao1 2 Qianqian Wu1 Jianqin Huang3 Chuan-Qing Zhang1 † 1Department of Plant Pathology, Zhejiang Agriculture and Forestry University, Hangzhou 311300, China 2Fujian University Key Laboratory for Plant-Microbe Interaction, College of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou, 350002, China 3State Key Laboratory of Subtropical Silviculture, Zhejiang Agriculture and Forestry University, Hangzhou, 311300, China Published Online:8 Mar 2022https://doi.org/10.1094/PDIS-08-21-1623-AAboutSectionsPDF ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat Genome AnnouncementBotryosphaeria dothidea is a latent pathogen with global importance to woody plant health, which causes serious tree trunk cankers on Chinese hickory. To date, only one Illumina short-read-based genome assembly of strain CK16 is available for host Chinese hickory. To address this problem, we report a near telomere-to-telomere genome assembly of strain BDLA16-7 using Oxford Nanopore Technologies (ONT) sequencing. The almost chromosomal-level and well-annotated genome assembly will provide a valuable genetic resource for understanding of the infection mechanisms of B. dothidea in future.Chinese hickory (Carya cathayensis Sarg.) is an endemic subtropical tree species of commercial importance, which is known for its distinctive fragrance and the high nutritional value of its nuts. However, nearly 90% of the Chinese hickory trees have been severely threatened by trunk cankers caused by B. dothidea (Zhang and Xu 2011). This disease occurs during late March to September with obvious latent infections, for which the pathogen primarily overwinters in the diseased trunks (Dai et al. 2017; Wang and Zhang 2019). Canker symptoms generally occur on trunks of hickory less than 2 m above the ground, and few lesions are observed on other parts such as twigs, branches, and nuts. B. dothidea is a worldwide fungal pathogen with global importance: damaging woody plants; infecting through wounds; breaching the outer bark and colonizing the phloem, vascular cambium, and xylem; and causing the weakening and decay of the woody hosts (Marsberg et al. 2017; Slippers et al. 2007).Currently, the genome sequences of six B. dothidea strains with host plants including apple (Liu et al. 2016; Wang et al. 2018; Yu et al. 2021), pear (Hu et al. 2019), kiwifruit (Liang et al. 2021), and Chinese hickory (Rao et al. 2021) are publicly available in the NCBI Assembly Database. The assembly sizes of the six strains were similar, ranging from 44.17 to 51.76 Mb (Table 1). Of these strains, only one stain, named CK16, was an agent for trunk canker disease on Chinese hickory (Rao et al. 2021). The genome assembly of CK16 was assembled based on Illumina short-read sequencing technology, and has the highest contig numbers among these strains.Table 1. Genome features of the Botryosphaeria dothidea strainsaFeaturesBDLA16-7CK16sdau11-99LW030101PG45LW-HubeiPTZ1HostChinese hickoryChinese hickoryAppleAppleApplePearKiwifruitSequencing technologyONT + IlluminaIlluminaPB + IllumIlluminaIlluminaPB + IllumPB + IllumReference (PubMed ID)This study3376177033258431277896353062288132647612Liang et al. 2021Assembly accessionbGWHBEBO00000000ASM1515935ASM1150312ASM171744ASM401626ASM1106463ASM1334390Assembly size (Mb)46.0544.3951.7647.3944.1746.3544.45Contig number151,303881,2514236828Contig N50 (Mb)3.870.524.040.210.352.722.68Contig L505266653777Average contig length (Mb)3.070.030.590.040.110.951.59Maximum contig length (Mb)6.191.486.151.261.154.863.62GC content (%)54.4554.6052.9353.0954.6054.3054.59Repeat sequence (%)c7.965.2913.9612.585.649.406.68BUSCO completeness (%)d99.0097.8397.1397.8397.8997.7197.82Protein-coding genes12,81213,85614,11810,41115,66114,09110,415BDLA16-7 unique genes−2611,7451,539194357246aAbbreviations: ONT = Oxford Nanopore Technologies, PB + Illum = PacBio + Illumina, and BUSCO = benchmarking universal single-copy orthologs.bAssembly of BDLA16-7 is available at the Genome Warehouse (GWH) (http://ngdc.cncb.ac.cn/gwh/) while other assembles are available at the NCBI Assembly database.cRepeats were conducted by RepeatMasker V4.1.2 with the de novo repeat library generated from assembly of BDLA16-7.dThe completeness of genome assemblies were assessed by BUSCO v5.1.2 at the Ascomycota level (n = 1,706).Table 1. Genome features of the Botryosphaeria dothidea strainsaView as image HTML Here, we isolated B. dothidea strain BDLA16-7 from the trunk of a Chinese 'Linan' hickory tree with canker disease in Linan, Zhejiang Province, China. Its colony was initially pale white on potato dextrose agar (PDA) plates, then became black. The pycnidia were typically solitary, gray-black, globular, and covered by mycelia. Its genomic DNA and messenger RNA were extracted from 10-day-old mycelium cultivated on PDA media. Its genome and transcriptome were sequenced by the ONT PromethION sequencing platform and Illumina HiSeq4000 sequencing platform, respectively, at Biomarker Technologies Co., Ltd. (Beijing, China). Finally, we obtained 5.89-Gb ONT long reads (approximately 108×, N50 20.41 kb) for genome assembling and 7.06-Gb Illumina short reads (approximately 156×, 2 × 150 bp) for genome assembly polishing and gene annotation (Table 1).The de novo genome assembler suit NextDenovo v2.4.0 and NextPolish v1.3.1 (both developed by NextOmics; https://github.com/Nextomics) were employed to generate a genome assembly of high accuracy and continuity. First, a draft genome assembly was assembled by NextDenovo using ONT long reads (seed reads ≥ 29,488 bp); then, it was polished by NextPolish using both ONT long reads and Illumina short reads. Finally, we obtained a 46.05-Mb genome assembly (GC content 54.45%) consisting of 15 contigs with a contig N50 of 3.87 Mb (L50 = 5). The average contig length was 3.07 Mb and the maximum contig length was 6.19 Mb (Table 1). Compared with the previously reported genome assembly of strain CK16, genome assembly of BDLA16-7 was a little bigger in size (46.05 versus 44.39 Mb), reduced 98.85% (15 versus 1,302) in number of contigs, and, thus, increased approximately 100-fold in average contig length (3.07 versus 0.03 Mb), approximately 7-fold (3.87 versus 0.52 Mb) in contig N50, and approximately-4 fold (6.19 versus 1.48 Mb) in the maximum contig length (Table 1). Compared with the genome assemblies of other host strains, the genome assembly of BDLA16-7 had the minimum contig number and the largest contig N50, as well as the maximum contig length (Table 1).The completeness of genome assembly BDLA16-7 was assessed by benchmarking universal single-copy orthologs (BUSCO v5.12; https://busco.ezlab.org/) at Fungi and Ascomycota levels. It contained 753 (99.34%) complete (749 single and 4 duplicated), 3 fragmented, and 2 missing orthologs at the Fungi level (n = 758), and 1,689 (99.00%) complete (1,678 single and 11 duplicated), 4 fragmented, and 13 missing orthologs at Ascomycota level (n = 1,706) (Fig. 1; Table 1). Compared with the BUSCO assessment results of other reported genome assemblies at the Ascomycota level, the completeness of genome assembly BDLA16-7 was the best one (Table 1). Also, the genome assembly completeness was valued by telomere repeats (5′-TTAGGG-3′ and 5′-CCCTAA-3′) at the start and end of contigs. Of 15 contigs, 8 and 6 contigs were found at the start or end, respectively, with telomere repeats, and 3 contigs had both ends with telomere repeats, which indicated that these contigs were reached at the telomere-to-telomere (Miga et al. 2020) chromosomal level.Fig. 1. Completeness of genome assembly and predicted genes were assessed by benchmarking universal single-copy orthologs (BUSCO v5.1.2) at the Fungi and Ascomycota levels.Download as PowerPointRepeats were masked before gene prediction by RepeatMasker v4.1.2 (http://www.repeatmasker.org/) with a de novo repeat library generated by RepeatModeler v2.01 (http://www.repeatmasker.org/RepeatModeler/). In total, 7.96% of repeats (mainly including approximately 3% long terminal repeat and approximately 2% long interspersed retrotransposable element) were identified in the genome assembly of BDLA16-7. It is a little higher than repeats of strains CK16 (5.29%), PG45 (5.64%), and PTZ1 (6.68%) but lower than repeats of strains sdau11-99 (13.96%), LW030101 (12.58%), and LW-Hubei (9.40%) (Table 1). The repeat-masked genome assembly of BDLA16-7 was used for gene prediction, and identified 12,812 protein-coding genes by BRAKER2 (Brůna et al. 2021), which integrated ab initio gene prediction from AUGUSTUS v3.4.0 (Stanke et al. 2008), as well as evidence from RNA-sequencing data and fungal homologous proteins (fungi_odb10) (https://busco-data.ezlab.org/v5/data/lineages/). Compared with other strains, the number of protein-coding genes of BDLA16-7 was in the middle (12,815 versus approximately 10,411 to 15,661), which may have been caused by different gene prediction methods. Using BLASTn search against other genome assemblies, the number of isolate-specific genes in BDLA16-7 ranged from 194 to 1,745 (Table 1). The BUSCO gene completeness assessment showed that the predicted gene set of BDLA16-7 included 739 (97.50%) and 1,650 (96.72%) complete orthologs at the Fungi and Ascomycota levels, respectively (Fig. 1).The gene functional annotation was conducted by InterProScan v5.51-85.0 (https://github.com/ebi-pf-team/interproscan) and eggNOG-mapper v2 (http://eggnog-mapper.embl.de/). In total, 10,272 (80.16%) genes were assigned a annotation from either Pfam (9,231), gene ontology (4,027), Kyoto Encyclopedia of Genes and Genomes (4,741), or eukaryotic orthologous groups (9,998). We identified a set of pathogenicity-related genes, including 3,642 pathogen–host interaction genes (PHI-base v4.12; http://www.phi-base.org/), 250 carbohydrate active enzymes (dbCAN2; https://bcb.unl.edu/dbCAN2/), and 252 cytochrome P450 enzymes. Furthermore, 752 putative secreted proteins were identified by SignalP v5.0 (Almagro Armenteros et al. 2019) and TMHMM v2.0 (http://www.cbs.dtu.dk/services/TMHMM/), following our previous method (with signal peptide but without any transmembrane domain in mature protein) (Bao et al. 2017).Fungal secondary metabolites such as fumonisins and deoxynivalenol were toxic to plants and, thus, played an important role in fungal infection and plant disease development (Diamond et al. 2013; Iqbal et al. 2021). The genes related to products of any secondary metabolite were fused as secondary metabolite biosynthesis gene clusters (SMBGCs) (Keller 2019). 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Wu contributed equally to this work.Funding: This work was supported by the grants from the Key Research and Development Project of Zhejiang Province, China (2020C02005), and Natural Science Foundation of Fujian Province, China (2019J01385).The author(s) declare no conflict of interest.DetailsFiguresLiterature CitedRelated Vol. 106, No. 3 March 2022SubscribeISSN:0191-2917e-ISSN:1943-7692 Download Metrics Downloaded 492 times Article History Issue Date: 30 Mar 2022Published: 8 Mar 2022First Look: 4 Nov 2021Accepted: 29 Oct 2021 Pages: 1023-1026 Information© 2022 The American Phytopathological SocietyFundingKey Research and Development Project of Zhejiang Province, ChinaGrant/Award Number: 2020C02005Natural Science Foundation of Fujian Province, ChinaGrant/Award Number: 2019J01385KeywordsBotryosphaeria dothideaChinese hickorygenometrunk canker diseaseThe author(s) declare no conflict of interest.PDF download