Scientia Agricultura Sinica ›› 2021, Vol. 54 ›› Issue (6): 1288-1300.doi: 10.3864/j.issn.0578-1752.2021.06.018

• ANIMAL SCIENCE·VETERINARY SCIENCE·RESOURCE INSECT • Previous Articles    

Improvement of Nosema ceranae Genome Annotation Based on Nanopore Full-Length Transcriptome Data

HuaZhi CHEN1(),YuanChan FAN1(),HaiBin JIANG1,Jie WANG1,XiaoXue FAN1,ZhiWei ZHU1,Qi LONG1,ZongBing CAI1,YanZhen ZHENG1,ZhongMin FU1,2,GuoJun XU1,DaFu CHEN1,2,Rui GUO1,2()   

  1. 1College of Animal Sciences (College of Bee Science), Fujian Agriculture and Forestry University, Fuzhou 350002
    2Apitherapy Research Institute, Fujian Agriculture and Forestry University, Fuzhou 350002
  • Received:2020-05-06 Accepted:2020-05-28 Online:2021-03-16 Published:2021-03-25
  • Contact: Rui GUO E-mail:CHZ0720@outlook.com;fanyc19980201@126.com;ruiguo@fafu.edu.cn

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Abstract:

【Objective】The objective of this study is to improve gene sequence and functional annotation of current reference genome of Nosema ceranae using previously obtained Nanopore full-length transcriptome dataset. 【Method】TransDecoder software was used to predict open reading frames (ORFs) of N. ceranae and corresponding amid acids. Comparison between full-length transcripts and transcripts annotated in reference genome was performed using gffcompare software to extend upstream sequences or downstream sequences of annotated genes’ untranslated regions and correct genes’ boundaries. MISA software was used to explore simple sequence repeat (SSR) loci within transcripts with a length above 500 bp, including single nucleotide repeat, dinucleotide repeat, trinucleotide repeat, tetranucleotide repeat, pentanucleotide repeat, hexanucleotide repeat and mixed SSR. By using Blast tool, novel genes and novel transcripts were aligned to Nr, KOG, eggNOG, GO and KEGG databases to gain functional annotations. 【Result】A total of 2 353 complete ORFs were predicted, and those ORFs with a length distribution among 0-100 aa were the predominant, reaching a ratio of 72.12% among total ORFs. Additionally, structures of 2 340 N. ceranae genes were optimized; 5′ ends of 1 182 genes and 3′ ends of 1 158 genes were respectively prolonged. Moreover, 1 658 SSRs were identified, and the numbers of single nucleotide repeat, dinucleotide repeat, trinucleotide repeat, tetranucleotide repeat were 1 622, 23, seven and six, respectively. The density of single nucleotide repeat was the highest (182.32/Mb), followed by those of mixed SSR, dinucleotide repeat and trinucleotide repeat, reaching 6.90, 2.78 and 0.73/Mb, respectively. Further, 954 novel genes were identified, among them 951, 333, 371, 422 and 321 were respectively annotated to Nr, KOG, eggNOG, GO and KEGG databases. In addition, 6 164 novel transcripts were identified, among them 6 141, 2 808, 2 932, 3 196 and 2 585 were annotated to the aforementioned five databases, respectively. The species annotated by the highest number of new gene and new transcript was N. ceranae followed by Nosema apis. 【Conclusion】Our results well improve sequences and functional annotations of annotated genes in current reference genome of N. ceranae, and supplement and annotate a number of unannotated novel genes and transcripts. Lots of SSR sites were provided for research on molecular markers, information of genes and transcripts on reference genome were supplemented.

Key words: Nanopore sequencing, full-length transcript, transcriptome, genome, honeybee, Nosema ceranae

Table 1

Summary of structural optimization of 10 genes in N. ceranae reference genome"

基因ID
Gene ID		 基因位置
Gene locus		 正负链
Plus and minus strand		 末端
End		 原位置
Original site		 优化后位置
Optimized site
Gene1175 NW_020169317.1:49652-50638 - 3′ 50341 50638
Gene326 NW_020169413.1:1478-3041 - 3′ 2683 3041
Gene503 NW_020169431.1:1208-2193 - 5′ 1461 1208
Gene503 NW_020169431.1:1208-2193 - 3′ 1634 2193
Gene1591 NW_020169298.1:96744-98975 + 5′ 96746 96744
Gene1591 NW_020169298.1:96744-98975 + 3′ 98671 98975
Gene588 NW_020169310.1:2436-3030 + 3′ 2741 3030
Gene350 NW_020169307.1:13073-18338 - 5′ 17277 13073
Gene36 NW_020169296.1: 54153-55245 - 5′ 54289 54153
Gene2363 NW_020169300.1:40046-41314 - 5′ 40510 40046

Table 2

Summary of searching result of N. ceranae SSRs based on MISA"

MISA搜索项目 MISA searching item 数量 Number
搜索基因 Searching gene 8021
搜索基因的总序列长度
Total sequence length of searching gene (bp)		 8265494
鉴定到的SSR位点 Identified SSR loci 1658
鉴定到的SSR总序列长度
Total sequence length of identified SSRs (bp)		 1405
含有1个以上SSR的基因
Genes containing more than one SSR		 212
混合SSR Mixed SSR 65
单核苷酸重复Single nucleotide repeat 1622
双核苷酸重复Dinucleotide repeat 23
三核苷酸重复Trinucleotide repeat 7
四核苷酸重复Tetranucleotide repeat 6

Fig. 1

Nr (A), KOG (B) and eggNOG (C) database annotations of novel genes of N. ceranae"

Fig. 2

GO database annotation of novel genes of N. ceranae"

Fig. 3

KEGG database annotation of novel genes of N. ceranae"

Fig. 4

Nr (A), KOG (B) and eggNOG (C) database annotations of novel transcripts of N. ceranae"

Fig. 5

GO database annotation of novel transcripts of N. ceranae"

Fig. 6

KEGG database annotations of novel transcripts of N. ceranae"

Fig. 7

Length distribution of amino acids encoded by complete ORFs of N. ceranae"

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