Deciphering the mitochondrial genome of Juglans mandshurica (Juglandaceae)

Abstract

Juglans mandshurica Maxim., 1856 is a second-class, protected, rare tree species of high economic and ecological value. We elucidated the complete mitochondrial (mt) genome of J. mandshurica using the Illumina Novaseq 6000 and Nanopore platforms. The complete sequences of 558,032 and 161,386 bp had an overall GC content of 45.0% and 45.3%, respectively, and 61 genes could be annotated, including 38 protein-coding, 20 tRNA, and 3 rRNA genes. The high-quality J. mandshurica mt genomic sequences presented in this study will serve as a useful resource for a range of genetic, functional, evolutionary, and comparative genomic studies on this species of the Juglandaceae family.

Keywords:

• Juglans mandshurica
• mitochondrial genome
• phylogenetic analysis

Introduction

Juglans mandshurica Maxim. is a second-class, protected, rare tree species that belongs to the genus Juglans of the Juglandaceae family (Wang et al. 2010). It is primarily scattered in mixed forest regions of northeastern China, northern Korea, and far eastern Russia, at altitudes of 400–1000 m (Chen et al. 2018; Liu et al. 2020). J. mandshurica has high ecological and economic value and is widely used as a source of wood, food, and medicine (Huang et al. 2010; Shen et al. 2015; Luo et al. 2017). Notably, it is a straight-root tree with strong tolerance to cold and drought that can survive at temperatures as low as −50 °C. Consequently, it is often employed as the parent plant for breeding cold-resistant Juglans regia (Liu 2020).

Plant mitochondrial (mt) genomes tend to have a more heterogeneous composition than chloroplast (cp) genomes (Zhang et al. 2019). Owing to the conservation of their genomic structure and a fairly low recombination rate, cp genomes provide valuable information for taxonomic classification and phylogenetic reconstruction, which aid in the identification of complex evolutionary relationships (Jansen et al. 2008; Parks et al. 2009; Zhang et al. 2019). In contrast, it is well recognized that the highly dynamic mt genome structure can have a variety of genomic conformations owing to recombination and interference based on repeated content (Palmer and Herbon 1988; Marechal and Brisson 2010). These characteristics make the plant mt genome a fascinating study topic, and as of March 24, 2022, as many as 437 plant mt sequences have been published (https://www.ncbi.nlm.nih.gov/genome/browse#!/organelles/). Although many studies have concentrated on the ecological characteristics, cultivation techniques, and chemical composition of J. mandshurica, there are very few exploring its genome (Wang et al. 2018; Pang, 2021; Liu et al. 2021). To our knowledge, the present study is the first report of the J. mandshurica mt genome.

Materials and methods

Plant materials and sequencing

Fresh J. mandshurica leaves were collected in July 2021 from the Forestry Experimental Station (37.52°N, 121.34°E) at Ludong University, Yantai City, China. Plant specimens (barcode number SDF1000958) and total genomic DNA (code number htq2021cp10) were stored at the Shandong Provincial Center of Forest and Grass Germplasm Resources (Biao Han, [email protected]). Total DNA was obtained using the Blood/Cell/Tissue Genomic DNA Extraction Kit (TIANamp Genoic DNA Kit, Tiangen, Beijing, China) (Uddin et al. 2014). The DNA library was constructed using the Ligation Sequencing Kit (SQK-LSK109) and sequenced using the Nanopore PromethION sequencing platform (Nanodrop Technologies, Wilmington, DE, US); 11.54 Gb raw data with an N50 length of 13,783 bp was generated. The data was filtered and re-edited using NanoFilt and NanoPlot in the Nanopack software (De Coster et al. 2018), resulting in 10.71 G clean reads. Libraries with an average fragment length of 350 bp were constructed; they were sequenced using the Illumina Novaseq 6000 platform (Illumina, San Diego, CA, USA) and 8.83 Gb raw data was generated. After editing the data using the NGS QC ToolKit v2.3.3 software (Patel and Jain, 2012), 8.78 Gb of data was generated from 29.28 million reads. The read coverage depth of Illumina and Nanopore sequencing exceeded 100×, indicating good sequencing data results (Figure S1).

Figure 1. Morphological characteristics of Juglans mandshurica. (a) whole plant, (b) fruits, (c) inflorescence, (d) seeds, (e) leaves.

Genome assembly and annotation

We first obtained a rough but computationally efficient assembly using the Miniasm tool (Li, 2016) after trimming adapter sequences with the Porechop software (https://github.com/rrwick/Porechop) and polished the resulting assembly with the Racon tool (https://github.com/isovic/racon). We selected contigs with homology to Fagus sylvatica (NCBI Reference Sequence: MT446430.1) mt genome using the Bandage software (Wick et al. 2015), retaining contigs with at least one ≥5 kb alignment to the F. sylvatica mitochondrion using the BLASTn tool (Chen et al. 2015). We then proceeded to align the Nanopore reads to our draft F. sylvatica assembly with the minimap2 tool (Li, 2018), segregated aligned reads, and assembled them de novo first using the Unicycler software (Wick et al. 2017) and then with the Flye software (Kolmogorov et al. 2019), as described previously. The final genome sequence was obtained by polishing with the Pilon tool using Illumina Novaseq 6000 sequencing reads. The mt genomes were annotated using the BLASTn, MITOFY (Alverson et al. 2010), and MFannot tools (http://megasun.bch.umontreal.ca/cgi-bin/mfannot/) and angiosperm mt genes were used to query sequences in the NCBI database (https://www.blast.ncbi.nlm.nih.gov). The tRNA genes were identified using the tRNA scan-SE software (http://lowelab.ucsc.edu/tRNAscan-SE/). The genome circle map of mt genome was visualized with the OGDRAW v1.2 software (http://ogdraw. mpimp-golm.mpg.de/). Manual correction was performed after software annotation to ensure the accuracy of annotation.

Phylogenetic analysis

The phylogeny of J. mandshurica was analyzed by aligning the mt genomic sequences of 18 species using the HomBlocks software (Bi et al. 2018) and subsequent trimming using the Gblock method. The Model-finder software was employed to chop the model WAG + I + G, followed by construction of the maximum likelihood (ML) phylogenetic tree using the RAxML v8.2.9 software with 1,000 bootstrap replicates (Stamatakis, 2014).

Results

Morphological characteristics

We obtained many photographs while observing and recording the morphological characteristics of J. mandshurica, including photographs of the whole plant, fruits, inflorescence, seeds, and leaves (Figure 1). J. mandshurica is an arbor with imparipinnate leaves. The fruits are globose, ovate, or elliptic. Flowering occurs in May, and the fruit stage is in August–September.

Genome sequencing assembly and characterization

Ample evidence suggests that plant mt genomes can exist in multiple circular configurations and even noncircular forms owing to intramolecular recombination events mediated by repeats (Kozik et al. 2019; Zhang et al. 2019; Logacheva et al. 2020). We obtained two complete circular J. mandshurica mt genomic molecules (558,032 bp and 161,386 bp) from the de novo assembly of the filtered mt reads with overall GC contents of 45.0% and 45.3%, respectively. Circular genome maps were constructed using the OGDRAW software (Figure 2), and 61 total genes, including 38 protein-coding genes, 20 tRNA genes, and 3 rRNA genes, were subsequently annotated (Table 1). Among these, 38 protein-coding genes were found to contain exons: atp1, atp4, atp6, atp8, atp9, ccmB, ccmC, ccmFN, cob, cox1, cox3, matR, mttB, nad3, nad4L, nad6, nad9, rpl5, rpl10, rpl16, rps1, rps3, rps4, rps7, rps10, rps12, rps14, rps19, sdh3, and sdh4 with one exon; ccmFc, cox2, and rpl2 with two exons; nad4 with four exons; and nad1, nad2, nad5, and nad7 with five exons. Moreover, eight protein-coding genes were found to contain introns: ccmFc, cox2, and rpl2 with one intron; nad4 with three introns; and nad1, nad2, nad5, and nad7 with four introns. Notably, nad1, nad2, and nad5 were found to undergo trans-splicing.

Figure 2. J. mandshurica mitochondrial (mt) genome gene map. The gene map denotes annotated genes based on different functional groups, which are color-coded on the outer circle as transcribed clock-wise (outside) and counter clock-wise (inside). The inner circle indicates the GC content as a dark grey plot.

Table 1. Gene composition of the J. mandshurica mt genome.

Group of genes	Name of genes
	Molecule 1	Molecule 2
Complex I	nad1, nad2, nad3, nad4, nad4L, nad5, nad6, nad9	nad1, nad7
Complex II	sdh3, sdh4
Complex III	ccmFc, ccmB, ccmC	ccmFN
Complex IV	cox1, cox2, cox3
ATP synthase	atp1, atp4, atp6, atp8, atp9(×2)
Cytochrome c biogenesis	cob
Maturases		matR
Transport membrane protein	mttB
Large subunit of ribosome	rpl2, rpl5, rpl10, rpl16
Small subunit of ribosome	rps3, rps4(×2), rps7(×2), rps10, rps12(×2), rps14, rps19	rps1, rps7
Ribosome RNA	rrnL	rrn5, rns
Transfer RNA	trnK(ttt), trnH(gtg), trnE(ttc)(×2), trnC(gca)(×2), trnP(tgg)(×2), trnW(cca), trnI(cat)(×2), trnD(gtc)(×2), trnS(tga)(×3), trnV(gac)(×2), trnL(caa), trnN(gtt), trnY(gta), trnL(aac), trnQ(ttg), trnG(gcc), trnM(cat)(×2), trnS(gct), trnF(gaa), trnR(acg)

The number in parentheses represents the number of copies of the gene, e.g. (×2) means that there are two copies.

Phylogenetic analysis

Phylogenetic analysis was performed using the ML method, based on the mt genome sequences of 19 species: including Eriobotrya japonica, Pyrus betulifolia, Prunus avium, Cannabis sativa, Morus notabilis, Hemiptelea davidii, Leucaena trichandra, Gleditsia sinensis, Robinia pseudoacacia, Dalbergia odorifera, Populus tremula, Salix paraflabellaris, Euonymus alatus, Cucurbita pepo, Citrullus lanatus, Luffa acutangula, Quercus variabilis, F. sylvatica, and Pinus Taeda. Our results revealed that J. mandshurica is most closely related to Q. variabilis and F. sylvatica (Figure 3).

Figure 3. Maximum likelihood (ML) phylogenetic tree of J. mandshurica and 19 other species based on the conserved segments of mitogenome sequences, with Pinus taeda as the outgroup. The accession numbers are showed in the figure, and the numbers behind each node are bootstrap support values.

Discussion and conclusion

Juglans L. is an economically and ecologically important genus of the Juglandaceae family (Hu et al. 2016). A total of five species (J. regia, J. sigillata, J. cathayensis, J. hopeiensis, and J. mandshurica) and one variety (J. draconia) belonging to this genus are found in China. The Juglans L. cp genome is highly conserved, with a typical four-segmented structure (LSC, SSC, IRa, IRb) (She, 2021). In contrast, mt genomes are highly diverse, which is evident based on the different plant mitogenome sequences that have been reported (Kozik et al. 2019; Liu et al. 2022). Although plant mitogenomes are often assembled and displayed as circular maps, they mostly exist as complex and dynamic collections of linear DNA interspersed with smaller circular and branched configurations (Oldenburg and Bendich et al. 2015; Morley and Nielsen 2017; Wynn and Christensen 2018; Kozik et al. 2019). For example, the Quercus acutissima mt genome is a branched structure comprising one linear and two circular formations (Liu et al. 2022), the Abelmoschus esculentus mt genome is composed of one linear and one circular structure (Li et al. 2022), and the Ipomoea batatas mt genome is arranged as four circular sections (Yang et al. 2022). The present study demonstrates that the J. mandshurica mt genome comprises two circular formations (558,032 bp and 161,386 bp); however, the coexistence of these molecules requires further investigation.

Given the low substitution rate in the mt genome, the constituent genes are a valuable source of information for phylogenetic analysis at high taxonomic levels (Hiesel et al. 1994). Our analysis of the phylogenetic relationships of J. mandshurica based on its mitochondrial genomic information revealed its close relationship with Q. variabilis and F. sylvatica. The high-quality mt genomic sequences presented in this study will serve as an important resource for a range of genetic, functional, evolutionary, and comparative genomic studies on J. mandshurica and other Juglans species of the Juglandaceae family.

Ethical approval

The authors complied with the international Union for Conservation of Nature (IUCN) policies research involving species at risk of extinction (see Guidelines for appropriate uses of IUCN Red List data), the Convention on Biological Diversity and the Convention on Trade in Endangered Species of Wild Fauna and Flora.

Author contributions

Xun Su (Data analysis and Thesis writing); Qiong Liu (Data analysis and Paper revision); Haili Guo (Data analysis and Thesis writing); Dechang Hu (Experimental design); Dan Liu (Experimental design and Paper revision); Zihao Wang (Resource investigation and Data interpretation); Ping Zhang (Data interpretation), and that all authors agree to be accountable for all aspects of the work.

Disclosure statement

The authors declare no conflict of interest.

Data availability statement

The genome sequence data are openly available in GenBank of NCBI at (https://www.ncbi.nlm.nih.gov/) under the accession no. MZ900993 and MZ900994. The associated BioProject, SRA, and Bio-Sample numbers are PRJNA757787, SRR15615977, SRR15615978, and SAMN20981794 respectively.

Additional information

Funding

The study was financially supported by Project of Shandong Natural Science Foundation Committee ‘Study on Fragmented Population Characteristics and Genetic Structure of Juglans mandshurica, a National Class II Endangered Plant, in Shandong Province’ (No. ZR2019MC055), the Subject of Key R & D Plan of Shandong Province (Major Scientific and Technological Innovation Project) ‘Mining and Accurate Identification of Forest Tree Germplasm Resources’ (No. 2021LZGC023), Shandong agricultural seed improvement project ‘Collection, preservation and precise identification of germplasm resources of characteristic ecological tree species’ (No. 2019LZGC01805), General survey of herbaceous plant germplasm resources in Shandong Province (No. LCYZ202101), and Wild Plant Conservation and Management Project of ‘Preservation of Germplasm Resources of Chinese Endemic Species of Wild Plants under National Key Protection’ (No. 2019073031).