The Complete Mitochondrial Genome Sequence Variation and Phylogenetic Analysis of Mulberry

doi:10.21203/rs.3.rs-445726/v1

Mulberry is admired for its landscaping and possesses high development prospects and scientific research value. Mitochondria are the plants' powerhouse that produces energy to carry out life processes. In this study, the mt genome of Morus L(M. atropurpurea and M. multicaulis)were annotated and assembled. The circular mt genome of M. multicaulis has a length of 361,546bp, contains 54 genes, including 31 protein-coding genes, 20 tRNA genes, and 3 rRNA genes and composition of A(27.38%), T (27.20%), C (22.63%) and G (22.79%). The sequence repeats, RNA editing gene and migration from cp to mt and was observed in M. multicaulis mt genome. Phylogenetic analysis based on the complete mt genomes of Morus and other 28 species reflects an exact evolutionary and taxonomic status.

Furthermore, we investigation on mt genome size, organization, and plastomes at the global level and pi analysis of Morus genome was investigated and compared to other land plants. The results indicate that the exist mt genome's variation in plants. We reported the mt genome assembly and annotation of a halophytic model plant, M. multicaulis, and subsequent analysis, which provided us with a comprehensive understanding of the Morus mt genome.

General Cell Biology & Physiology

Molecular Genetics

M. multicaulis，M. atropurpurea

Mitochondrial genome

variation，Phylogenetic analysis

Morus is an economically significant crop and strong saline-alkali tolerance belonging to the Moraceae family which is native to China and also been planted in various areas for erosion control and windbreaks all over the world (He N, 2013). Great value as a food for healthy M. alba also can be used as a herbal medicine to cure fever, improve eyesight, strengthen joints, and lower blood pressure in China(Chan et al., 2016).

The mitochondrial(mt) genome is power source for energy synthesis and conversion, providing energy protection for various physiological activities of cells(Kozik, Rowan, Lavelle, Berke, & Christensen, 2019) such as cell differentiation, apoptosis,cell growth and cell division(Rehman et al., 2012).In addition,it is also involved the synthesis and degradation of several compounds(Shtolz, Dan, & Evolution, 2019),therefore, mitochondrial play an essential role in plant productivity and development (Yasunari et al., 2005).The mt genome with highly conserved, but the mt genomes has significant differences in length,gene sequence and content(Richardson, Rice, Young, Alverson, & Palmer, 2013).The smallest known terrestrial plant is about 66 Kb,and the largest terrestrial plant mt genome length is 11.3 Mb(Daniel et al., 2012; Skippington, Barkman, Rice, & Palmer, 2015),mostly vary from 200 kb to 3Mb and larger than mt genomes of other eukaryotes(X. & Physiology, 2006).The mt genome structures are shaped by active recombination, gene transfer to the nucleus, and other forces that remain unclear show that by Physical mapping and sequencing of some of the small mt genomes(Woloszynska, 2009).Structural analyses revealed high frequencies of intra- and intermolecular recombination,which generated a structurally dynamic assemblage of genome configurations(Alverson et al., 2010).The mt genome are inherited from the maternal parent(Wolfe, Li, & Sharp, 1988),this provides a powerful model for the study of genome structure and evolution,also a certain advantages in phylogenetic reconstruction.These genomes exhibit an intriguing mixture of conservative (slowest rates of nucleotide substitution)(Drouin, Daoud, Xia, & Evolution, 2008) and dynamic evolutionary patterns.Some previous reported(Tong, Kim, & Park, 2016) also suggested that for evolutionary studies it is not necessary to assemble whole organelle genomes but just exploring the variations.

Currently, With the rapid development of sequencing technology,an increasing number of complete plant mt genomeswere assembled and reported Up to Jan. 2021, 351 complete mt genomes have been deposited in GenBank Organelle Genome Resources(Cheng, He, Priyadarshani, Wang, & Qin, 2021).However,the mt genome of Morus is incomplete and unexplored..In this study, we sequenced and annotated the mt genome of cultivated Morus(M. atropurpurea and M. multicaulis) and compared it with the wild M. notabilis(NC-041177.1) and other eudiot which provides additional information for a better understanding of the genetics of the Morus L.

Plant material, DNA extraction, and sequencing

The M. atropurpurea and M. multicaulis plants were collected from Jiangsu University of Science and Technology Sericulture Research Institute.National Mulberry Genebank Zhenjiang, China affiliated to the Chinese Academy Of Agricultural Sciences.Materials used this time have been officially approved by Chinese Academy Of Agricultural Sciences.Plant Genomic DNA Kit was used to isolate total genomic DNA from 100 mg fresh leaves and DNA sample quality was examined with agarose-gel electrophoresis, and the concentration was measured by Nanodrop instrument then qualified samples were sent to the Oxford Nanopore PromethION for sequencing.

Assembly and annotation of the mitochondrial genome

The mt genome sequence of mulberry were selected using blast v2.6 (https://blast.ncbi.nlm.nih.gov) /Blast.cgi) align the contig with the plant mitochondrial gene database (the mitochondrial gene sequence of the species published on NCBI).Subsequently,assembled by the software Canu(Sergey et al., 2017) with the selected reads.Use NextPolish1.3.1 (https://github.com/Nextomics/NextPolish) to calibrate and pilon(Walker, 2015) correct read errors to get the final assembly results .The encoded protein and rRNA use blast to align the published plant mitochondrial sequence as a ref,and then make further manual adjustments according to relative species.TRNA is annotated with tRNAscanSE (http://lowelab.ucsc.edu/tRNAscan-SE/).ORF uses OpenReading Frame Finder (http://www.ncbi.nlm.nih.gov/gorf/gorf.html) for annotation. After checking and manually confirmed the final annotation result is obtained.Use OGDRAW (https://chlorobox.mpimp-golm.mpg.de/OGDraw.html) to drawn a circular mitochondrial genome map.

Analysis of repeated sequences

The software misa (1.0) was identification tool used to detect simple sequence repeats(Sebastian et al., 2017).The type of p1,p2, p3, p4, p5, and p6 refer to (1/8) (2/5) (3/3) (4/3) (5/3) (6/3) bases with (unit size / minimum number of repeats),were identified in this analysis. The scattered repetitive sequences were detected using vmatch v2.3.0(http://www.vmatch.de/) Combining Perl scripts to identify repetitive sequences.,with minimum length set to 30 bp and hamming distance was 3.

RNA editing analyses and chloroplast to mitochondrion DNA transformation

The editing sites in the mitochondrial RNA of M. multicaulis were use online sites to predictions (http://www.prepact.de/prepact-main.php). DNA migration is common in plants and varies from species to species occurs during autophagy, gametogenesis, and fertilization(Huang, Ayliffe, & Timmis, 2003).We using blast software set similarity to 70%, and E-value to 10E-5 use circos v0.69-5 to visualize it.The cpDNA of M. multicaulis(KU355297) was downloaded from NCBI Organelle Genome Resources Database

Variation architecture and Phylogenetic tree construction

Nucleic acid diversity(pi) by maft software (--auto mode) to compare the homologous gene sequences of different species globally, and use dnasp5 to calculate. Comparison of the mt genome sequence with other plastomes at the global level using mVISTA online software in shuffle-LAGAN mode.MEGA7.0 was used for phylogenetic tree construction by the maximum likelihood (ML) and neighbor-joining (NJ) methods with a bootstrap of 1,000 Poisson model .The mt genomes date were downloaded from NCBI, including M.notabilis(NC-041177.1)

Statement

The experimental materials for this time are only experimental research and no field research. They were collected from my own school,National Mulberry GeneBank Zhenjiang. This collection was supported by Chinese Academy Of Agricultural Sciences and with the guidance of the school official leaders.The collection was conducted under the conditions permitted by national laws and regulations, Strictly abide by relevant laws and get official permission.My corresponding author also made a statement. After the collection, it is only for experimental research and has no other purpose.

Genome content and organization

The M.multicaulis mt genome is circular was determined to be 361,546 bp long,base composition of the genome is A(27.38%), T (27.20%), C (22.63%), G (22.79%) contains 54 functional genes including 3 rRNA genes, 20 tRNA genes, and 31 PCGs Pseudogenes and ORFs were all non-coding(Table 1).The mt genome of M.multicaulis functional categorization and physical locations of the annotated genes were presented(Fig. 1),encodes 31 different protein that could be divided into 9 classes(Table 3): ATP Synthase(5 genes),Cytochrome C Biogenesis (4 genes), Ubiquinol Cytochrome c Reductase (1 gene),Cytochrome C oxidase(3 genes),Maturases (2 gene) ,Transport membrane protein (1 gene),NADH dehydrogenase (9 genes), Ribosomal proteins (SSU) (5 genes) and Ribosomal proteins (LSU) (1 gene).

The mt genome sequence of M.atropurpurea is also circular was to be found 395,412-bp long(Figure 2) base composed of C+G (45.50%) including 57 functional genes contains 2 rRNA genes, 22 tRNA genes and 32 PCGs,31 different protein slao can be divided into 9 classes(Table 2.)

Table 1

Comparison of mt genomes among four species of *Morus* L.
Characteristics	M. notabilis	M.multicaulis	M.atropurpurea
Size (bp)	362,069 bp	361546	395412
GC content (%)	45.66	45.42	45.50
Number of genes	54	54	57
Protein-coding genes	26	31	32
rRNA	3	3	3
tRNA	21	20	22

Variations and Codon usage

.In this study, themt genome of M. multicaulis and M. atropurpurea were encoded by 27,933 and 28,251 codons.For M. multicaulis 31 protein-coding genes in the mt genome were encoded by 27,933 codons respectively, 62.2% of codons end in A or T Leu accounts for the highest codon usage(3,084) followed by Ser(2,454) and Arg(1,824)(Fig. 3.).These three amino acids almost represent four fifths of the total codons.the least number of codons is Trp (459).All of the protein-coding genes used AUG(753) most common start codon and three stop codons UAA, UGA,and UAG with the following utilization rate:UAA(53.33%), UGA(23.33%), and UAG(23.33%), (Table 3). Interesting for M. atropurpurea the most high codon usage is Leu and follow by Ser and Arg(Fig. 4) .

Previous reported shown that the mt genomes contain variable number of introns(Xiaofang et al., 2018).In our results the mt genome of M. multicaulis has 8 intron-containing genes (ccmFC, cox2, nad1, nad2,nad4,nad5,nad7,trnF-AAA) harboring 21 introns in total and nad1,nad2,nad5,nad7 even contains 4 introns, which is the highest intron number.M. atropurpurea also have 8 intron-containing genes contain 21 introns.Most land plants contain 3rRNA genes(Archibald, 2011). Consistently, our two species three same rRNA genes rrn18,rrn26 and rrn5 were annotated in Morus mt genome.Besides, 20 different transfer RNAs were identified In M. multicaulis mt genome transporting 19 amino acids,which more than one transfer RNAs might transport the same amino acid with different codons(Table 3.).

Table 2

Genes present in the mt genome of *M. atropurpurea* and *M. multicaulis*.
Group of genes	M. multicaulis	M. atropurpurea
ATP synthase	atp1 atp4 atp6 atp8 atp9	atp1 atp4 atp6 atp8 atp9
Cytohrome c biogenesis	ccmB ccmC ccmFC* ccmFN	ccmB ccmC ccmFC* ccmFN
Ubichinol cytochrome creductase	cob	Cob
Cytochrome c oxidase	cox1 cox2* cox3	cox1 cox2* cox3
Maturases	matR(2)	matR
Transport membrance protein	mttB	mttB
NADH dehydrogenase	nad1** nad2 nad3 nad4 nad4L nad5** nad6 nad7** nad9	nad1** nad2 nad3 nad4 nad4L nad5** nad6 nad7** nad9
Ribosomal proteins (LSU)	rpl16	rpl16
Ribosomal proteins (SSU)	rps12 rps19 rps3 rps4 rps7	rps12 rps13 rps19(2) rps3 rps4 rps7
Succinate dehydrogenase	ψsdh4	ψsdh4
Ribosomal RNAs	rrn18 rrn26 rrn5	rrn18 rrn26 rrn5
Transfer RNAs	trnC-GCA trnD-GTC trnE-TTC trnF-AAA* trnF-GAA trnK-TTT trnL-CAA trnM-CAT(4) trnN-GTT trnP-TGG(2) trnQ-TTG(2) trnR-ACG trnS-TGA trnW-CCA trnY-GTA	trnA-TGC(2) trnC-GCA trnD-GTC trnE-TTC(2) trnF-AAA trnF-GAA trnK-TTT trnL-CAA trnM-CAT(4) trnN-GTT trnP-TGG(2) trnQ-TTG trnR-ACG trnS-TGA trnW-CCA trnY-GTA

Notes: The numbers after the gene names indicate the duplication number. “*” indicate genes containing one or more introns. “ψ” indicate pseudogene.

Table 3

Codon usage in *M. multicaulis* and *M. atropurpurea*( AA:Amino Acid).
Codon	AA	M. multicaulis		M. atropurpurea		Codon	A A	M. multicaulis		M. atropurpurea
Codon	AA	No.	RSCU	No.	RSCU	Codon	A A	No.	RSCU	No.	RSCU
UAA	Ter	16	1.5999	15	1.4517	AUG	Met	251	1	256	1.9922
UAG	Ter	7	0.6999	6	0.5805	AAC	Asn	101	0.6756	102	0.6868
UGA	Ter	7	0.6999	10	0.9678	AAU	Asn	198	1.3244	195	1.3132
GCA	Ala	148	0.9656	150	0.9676	CCA	Pro	135	1.0908	134	1.0828
GCC	Ala	135	0.8808	139	0.8968	CCC	Pro	103	0.8324	104	0.8404
GCG	Ala	75	0.4892	75	0.484	CCG	Pro	69	0.5576	69	0.5576
GCU	Ala	255	1.664	256	1.6516	CCU	Pro	188	1.5192	188	1.5192
UGC	Cys	52	0.7592	54	0.7714	CAA	Gln	198	1.5408	206	1.5488
UGU	Cys	85	1.2408	86	1.2286	CAG	Gln	59	0.4592	60	0.4512
GAC	Asp	100	0.6802	103	0.6822	AGA	Arg	138	1.362	138	1.3488
GAU	Asp	194	1.3198	199	1.3178	AGG	Arg	75	0.7404	78	0.762
GAA	Glu	277	1.406	286	1.4158	CGA	Arg	124	1.2234	125	1.2216
GAG	Glu	117	0.594	118	0.5842	CGC	Arg	66	0.6516	67	0.6546
UUC	Phe	263	0.858	262	0.8576	CGG	Arg	79	0.7794	77	0.7524
UUU	Phe	350	1.142	349	1.1424	CGU	Arg	126	1.2432	129	1.2606
GGA	Gly	231	1.4236	238	1.4448	AGC	Ser	81	0.594	82	0.5952
GGC	Gly	88	0.5424	87	0.528	AGU	Ser	145	1.0638	148	1.074
GGG	Gly	115	0.7088	116	0.704	UCA	Ser	157	1.1514	161	1.1682
GGU	Gly	215	1.3252	218	1.3232	UCC	Ser	137	1.005	137	0.9942
CAC	His	63	0.5338	64	0.529	UCG	Ser	106	0.7776	106	0.7692
CAU	His	173	1.4662	178	1.471	UCU	Ser	192	1.4082	193	1.4004
AUA	Ile	200	0.822	203	0.8208	ACA	Thr	118	0.9896	118	0.9792
AUC	Ile	206	0.8466	207	0.837	ACC	Thr	123	1.0316	123	1.0208
AUU	Ile	324	1.3314	332	1.3422	ACG	Thr	68	0.5704	68	0.5644
AAA	Lys	227	1.201	236	1.2164	ACU	Thr	168	1.4088	173	1.4356
AAG	Lys	151	0.799	152	0.7836	GUA	Val	171	1.1592	171	1.1516
CUA	Leu	169	0.9864	168	0.9768	GUC	Val	108	0.7324	109	0.734
CUC	Leu	96	0.5604	96	0.558	GUG	Val	124	0.8408	123	0.8284
CUG	Leu	101	0.5892	99	0.5754	GUU	Val	187	1.2676	191	1.286
CUU	Leu	213	1.2432	212	1.2324	UGG	Trp	153	1	152	1
UUA	Leu	248	1.4472	253	1.4712	UAC	Tyr	75	0.5154	75	0.512
UUG	Leu	201	1.173	204	1.1862	UAU	Tyr	216	1.4846	218	1.488
UUG	Met			1	0.0078

Repeat sequences analysis

Simple sequence repetitions (SSRs) refers to DNA fragments consisting of short units of sequence repetition of 1–6 base pairs in length(B et al., 2014).In this study,we presented the mt genome of M. multicaulis there are 386 SSRs were identified by software misa (1.0) and the proportion of different forms showed that SSRs in monomer and trimer was the most abundant types accounted for 86.12% of the total SSRs.There are only One each for pentamer and hexamer and our study also found 6 complex repeating type(Table 4) this phenomenon could contribution to further evolution and genome analyses.

Tandem repeats, also named satellite DNA, widely found in eukaryotic genomes and some prokaryotes(GAO H, 2005),whereas Scattered repetitive sequences are another type of repetitive sequences that different with tandem repetitive sequences, distributed in a dispersed manner in the genome.Use vmatch v2.3.0 software to identify as follows:forward, palindromic, reverse, complement. It is shown that the 30–40 bp repeats are most abundant for both species.In M. multicaulis mt genome there were 53 Scattered repetitive sequences and the longest repeat was 22,003 bp were longer than M. atropurpurea for 1072bp(Figs. 5 and 6).

The prediction of RNA editing

RNA editing that refers to the addition, loss and conversion of the exist in the coding region of the transcribed RNA(Brennicke, Marchfelder, & Binder, 1999)found in all eukaryotes and plants(Malek, Lättig, Hiesel, Brennicke, & Knoop, 1996) that, the conversion of specific cytosine into uridine can alters the genomic information has been reported(§ & Knoop, 2016).We use online sites to predictions (http://www.prepact.de/prepact-main.php),showed that total 377 RNA editing sites within 22 protein-coding genes were predicted while mttB,nad5 and ccmC have the most editing sites predicted(32) atp9 even creat start condon ,and remaining 8 protein-coding gene (atp1,atp6, atp8,cox1,cox2,cox3,rpl16,rps19) does not have any editing site predicted in the mt genome of M. multicaulis(Fig. 7).Among those editing sites, 63.93%(241) were located at the second base of the triplet pos.and36.07% (136) were occurred with the first position of the triplet pos.

In this analysis,the hydrophobicity of 42.18% of amino acids did not change. However, 9.28% of the amino acids were predicted to change from hydrophobic to hydrophilic and 48.54% were were predicted to change from hydrophilic to hydrophobic.The RNA editing might lead to the premature termination of protein-coding genes, and this phenomenon showed in Our results that theamino acids of predicted editing codons a leucine tendency after RNA editing(Table 5).

Table 4

Distribution of SSR Type in the mt genomes of *M. multicaulis*.
Type	SSR	size	NO.	SSR	size	NO.	SSR	size	NO.	SSR	size	NO.
p1	(A)10	10	16	(C)8	8	4	(G)9	9	1	(T)14	14	3
	(A)12	12	2	(C)9	9	1	(T)10	10	18	(T)15	15	1
	(A)8	8	28	(G)10	10	1	(T)11	11	2	(T)8	8	26
	(A)9	9	12	(G)11	11	1	(T)12	12	1	(T)9	9	18
	(C)11	11	2	(G)8	8	8
p2	(AG)5	10	4	(CT)5	10	2	(TA)5	10	1	(TC)5	10	2
p2	(AG)6	12	2	(CT)6	12	2
p3	(AAC)3	9	1	(ATT)3	9	3	(GAA)3	9	12	(GTT)4	12	2
	(AAC)4	12	2	(CAA)3	9	3	(GAC)3	9	2	(TAA)3	9	3
	(AAG)3	9	4	(CAC)3	9	4	(GAG)3	9	2	(TAC)3	9	1
	(AAG)4	12	2	(CAG)3	9	1	(GAT)4	12	1	(TAG)3	9	3
	(AAT)3	9	3	(CAT)3	9	2	(GCA)3	9	1	(TAT)3	9	2
	(ACC)3	9	2	(CCA)3	9	1	(GCC)3	9	3	(TCA)3	9	1
	(ACG)3	9	2	(CCG)3	9	2	(GCT)3	9	9	(TCC)3	9	5
	(ACT)3	9	2	(CCT)3	9	1	(GCT)4	12	1	(TCT)3	9	10
	(ACT)4	12	1	(CCT)4	12	1	(GGA)3	9	1	(TGA)3	9	1
	(AGA)3	9	11	(CGC)3	9	1	(GGC)3	9	1	(TGC)3	9	1
	(AGC)3	9	6	(CTA)3	9	4	(GGT)3	9	1	(TGT)3	9	1
	(AGG)3	9	4	(CTC)3	9	3	(GTA)3	9	2	(TTA)3	9	4
	(AGG)5	15	1	(CTG)3	9	3	(GTC)3	9	1	(TTC)3	9	10
	(AGT)3	9	1	(CTT)3	9	11	(GTG)3	9	1	(TTC)4	12	1
	(ATA)3	9	7	(CTT)4	12	1	(GTT)3	9	5	(TTC)5	15	1
	(ATC)3	9	3	(TTG)3	9	5
p4	(AAAG)3	12	1	(CAAA)3	12	1	(GGAA)3	12	1	(TGCT)3	12	1
	(AACA)3	12	1	(CCTT)3	12	1	(GTCA)3	12	1	(TTAT)3	12	1
	(AAGT)3	12	1	(CTTT)3	12	1	(TAAG)3	12	1	(TTCT)3	12	3
	(AATG)3	12	1	(GAAT)3	12	1	(TAGG)3	12	1	(TTGA)4	16	1
	(ACTC)3	12	1	(GACC)3	12	1	(TCGC)3	12	1	(TTTA)3	12	1
	(ATAA)3	12	1	(GCCG)3	12	1	(TCTT)3	12	1	(TTTC)3	12	2
	(ATCT)3	12	1	(GCTT)3	12	1	(TGAA)3	12	2
p5	(TGAGT)3	15	1	(TGAGT)3	15	1
p6	(AAGGAG)3	18	1	(GAAAGA)3	18	1
c	(A)11c(A)8	20	1	(T)9(G)9	18	1
c*	(CTA)3(TA)5*	17	1

Table 5

Prediction of RNA editing sites of *M.multicaulis*.mt genome
Type	Codon	Aa change	Number	Percentage
hydrophobic	TTT->CTT	F->L	4	28.12%
	TTG->CTG	F->L	3
	GCT->GTT	A->V	1
	GCG->GTG	A->V	2
	GCA->GTA	A->V	1
	CTT->TTT	L->F	11
	CTC->TTC	L->F	3
	CCT->CTT	P->L	20
	CCG->CTG	P->L	19
	CCC->CTC	P->L	8
	CTC->CCC	L->P	1
	CCA->CTA	P->L	33
hydrophilic	CGT->TGT	R->C	23	14.06%
	CGC->TGC	R->C	9
	CAT->TAT	H->Y	13
	CAC->TAC	H->Y	8
hydrophobic-hydrophilic	CCT->TCT	P->S	17	9.28%
	CCG->TCG	P->S	5
	CCC->TCC	P->S	11
	CCA->TCA	P->S	2
hydrophilic-hydrophobic	TCT->TTT	S->F	34	48.54%
	TCG->TTG	S->L	62
	TCA->TTA	S->L	49
	ACT->ATT	T->I	4
	ACG->ATG	T->M	3
	ACC->ATC	T->I	1
	ACA->ATA	T->I	3
	TCC->CCC	S->P	1
	TCA->CCA	S->P	2
	CGG->TGG	R->W	24

Homology analysis of chloroplast with mitochondria

DNA migration is common in plants(Chang et al., 2013).Homologous sequence between chloroplast and mitochondria found using blast software,set similarity to 70%, and E-value to 10E-5 use circos v0.69-5 to visualize it.Twenty-five fragments with a total length of 28,207bp were observed to be migrated from cp genome to mt genome in M.multicaulis., accounting for 7.80% of the mt genome(Fig. 8). There are 7 annotated genes located on those fragments, all of which are tRNA genes, namely trnL-CAA,trnN-GTT,trnM-CAT,trnP-TGG,trnW-CCA,trnD-GTC and trnM-CAT.Our data also demonstrate that some chloroplast protein-coding genes migrated from cp to mitochondrion, most of them lost their integrities during evolution, and only partial sequences of those genes could be found in the mt genome such nad1 ccmC, rrn18.

Table 6

Fragments transferred from chloroplast to mitochondria of *M.multicaulis*.mt genome
	length	identity	Mis match	Gap opens	mt start	mt end	cp start	cp end	Gene
1	3112	98.747	13	3	91,640	88,555	150,948	154,059
2	3112	98.747	13	3	88,555	91,640	93,119	96,230
3	2936	99.251	8	1	100,235	97,314	96,350	99,285
4	2936	99.251	8	1	97,314	100,235	147,893	150,828	trnL-CAA
5	2681	99.925	1	1	118,160	115,481	134,823	137,503	trnL-CAA
6	2681	99.925	1	1	115,481	118,160	109,675	112,355	trnN-GTT
7	2180	99.083	11	1	346,348	344,178	91,029	93,208	trnN-GTT
8	2180	99.083	11	1	344,178	346,348	153,970	156,149
9	1073	98.788	5	1	349,695	348,631	88,408	89,480
10	1073	98.788	5	1	348,631	349,695	157,698	158,770
11	521	87.716	18	9	7,055	7,529	796	1,316
12	235	100	0	0	221,648	221,414	89,864	90,098
13	235	100	0	0	221,414	221,648	157,080	157,314	nad1*,ccmC,trnM-CAT
14	889	74.241	174	42	152,321	151,463	141,980	142,843	nad1*,ccmC,trnM-CAT
15	889	74.241	174	42	151,463	152,321	104,335	105,198	rrn18
16	507	79.29	67	25	87,604	88,091	69,809	70,296	rrn18
17	166	100	0	0	84,192	84,027	104,835	105,000	trnP-TGG,trnW-CCA
18	166	100	0	0	84,027	84,192	142,178	142,343
19	156	92.308	7	2	122,916	123,067	59,360	59,514
20	148	92.568	10	1	245,879	245,732	36,734	36,880
21	82	97.561	1	1	39,222	39,142	32,340	32,421
22	79	94.937	4	0	141,020	140,942	55,104	55,182	trnD-GTC
23	62	90.323	4	2	164,557	164,498	146,669	146,730	trnM-CAT
24	62	90.323	4	2	164,498	164,557	100,448	100,509
25	46	95.652	0	1	326,639	326,596	45,875	45,920
Total	28,207

The different destinations of transferred protein-coding genes and tRNA genessuggested that tRNA genes are much more conserved in the mt genome than the protein-coding genes,indicating their indispensable roles in mitochondria.

Comparison with others green plant mt genomes

Comparison of the mulberry mt genome sequence with other plastomes at the global level using mVISTA online software in shuffle-LAGAN mode of Morus species with four family(Leguminosae,Gramineae,Rosaceae,Asteraceae). M.notabilis used as the reference in the comparative analysis.Interestingly, four family remarkably group-specific and each group shows nearly identical patterns among themselves.The vista plot patterns produced are remarkably group-specific and each group shows nearly identical patterns among themselves(Fig. 9).

Variation architecture at the mt genome level

Nucleic acid diversity(pi) can reveal the variation of nucleic acid sequences of different species, and regions with higher variability can provide potential molecular markers for population genetics.Use maft software (--auto mode) to compare the homologous gene sequences of different species globally, and use dnasp5 to calculate the pi value of each gene. In the mt genome the nucleotide diversity (pi) of the mt genome in cultivated species M. multicaulis with wild species of M.notabilis and was calculated.In our research found 10 gene(cox1,ccmFc,cob,ccmFN,nad9,mttB,nad3,nad4,atp4,atp9,rps3) pi ranged from 0.00063 to 0.02182 slide window among whole mt genome(Fig. 10). Most of the pi values were lower than 0.01,while rps3 accounting for highest with 0.02182.Besides,total, 85 variations including 79 SNPs and 6 indels were identified across the mt genomes of M. multicaulis and M. atropurpurea(Table 7).This phenomenon could be applied to further analyses of Morus mt genome evolution.

Table 7

Summary the total variations (SNPs and Indels) in *M. multicaulis* and *M. atropurpurea*.
Summary	Type	Total variation
	SNPs	79
	Indels	6
	Total	85

Phylogenetic analysis within Dicotyledon mt genomes

To understand the evolutionary status of Morus we use MEGA(7.0) to analysis moraceae together with others 7 dicotyledon total 28 species based on the complete mt genome sequence and construct the phylogenetic tree through the ML and NJ methods with a bootstrap of 1,000 replicates to assess the reliability.In this study we collect 28 eudicot from 8 families(Moraceae, Leguminosae, Gramineae, Brassicaceae, Malvaceae, Cucurbitaceae, Asteraceae. Solanaceae) were well clustered and showed that the phylogenetic tree strongly supports the order of taxa in the phylogenetic tree was consistent with the evolutionary relationships of those species, indicating theconsistency of traditional taxonomy with the molecular classification. Based on the phylogenetic relationships among the 28 species, different groups of plants can applied to further comparative analysis.For Moraceae two methods ML and NJ all showed grouped M. atropurpurea and M. multicaulis together. Which revealed that M. atropurpurea and M. multicaulis are more related to their congeners than to others and this analysis are important for the mt genome project, development of molecular markers for Morus species(Fig. 11 and12).

Mitochondria are the power source of the plants that produce the required energy to carry out life processes account of extensive size variations, sequence arrangements, repeat content and highly conserved coding sequence so possess more complex than animals(Kozik et al., 2019).We studied the characteristics of the mt genome of mulberry, a crucial salt tolerance and economically plant with great value as a food and medicine.It is reported that most of the mt genomeis is circular, and few are linear such as the mt genome of Polytomella parva in plants(Notsu et al., 2002; Smith, Lee, & Evolution, 2008).In this report, the mt genome of M. multicaulis M. atropurpurea has shown that is circular and respectively with 361,546bp and 395,412bp in size,and GC content of the mt genome Morus also supports the conclusion that GC content is highly conserved in higher plants.

The repeat sequences contain tandem, short, and large repeats are widely exist in the mt genome(Guo, Zhu, Fan, & Mower, 2016),that play a vital role in shaping the mt genome accounting for those repeats in mitochondria are pivotal for intermolecular recombination(Dong et al., 2018).In this study, we focus on reported the SSRs and scattered repetitive sequences intensively. Research has shown that M. multicaulis harbors abundant repeat sequences that might indicate that the intermolecular recombination frequently happens in the mt genome, which maybe applied to dynamically changes the sequence and conformation during the evolution.

RNA-editing is mean posttranscriptional process that occurs in the both cpDNA and mt genomes of higher plants, which contributing to the better folding of proteins(Bi et al., 2016).So identification of RNA-editing sites provides essential clues for future analysis of predicting gene functions with novel codons about evolution ,that can helps us better to understand the gene expression of the cpDNA and mt genomes in plants.Previous studies have been shown that Arabidopsis total 441 RNA-editing sites within 36 genes(Unseld, Marienfeld, Brandt, & Brennicke, 1997), rice have 491 RNA-editing sites within 34 genes(Notsu et al., 2002) and216 RNA-editing sites within 26 genes of S. glauca(Cheng et al., 2021).Our results show 377 RNA-editing sites within 22 protein-coding genes were predicted of M. multicaulis. The tRNA genes are much more conserved in the mt genome than the protein-coding genes,indicating their indispensable roles in mitochondria. As the cytoplasmic genome, migration of cpDNA to the mt genome occurred during the plant evolution. We found that 25fragments were transferred from the cp genome to mt with 7 integrated genes, which are all tRNA genes(Table 6). Transfer of tRNA genes from cp to mt is common in angiosperms(Bi et al., 2016).

We also investigated the mulberry plastome sequence with other land plants at the global level.Conclusively, the genome structure and organization of Morus were consistent together and have a significant differences with other terrestrial green plants.nucleotide diversity (pi) of the mt genome in M. multicaulis was calculated.In our research found 10 gene ranged from 0.00063 to 0.02182 maybe relative to the evolution.Further, we have analyzed the phylogenetic relationship of mulberry with eudicot representative taxa based on the complete mt genome sequence. Interestingly, Two cultivatedare more related than others which familiar to preious reported(QL, Guo, JZ, Yan, & RES, 2016).

In this study, we collected two cultivated species of Morus L.(M.atropurpurea and M. multicaulis) assembled and annotated the mt genome and performed extensive analyses based on the complete mt genome sequences and amino acid sequences of the annotated genes. The Morus L mt genome is circular, M. multicaulis with a length of 361,546 bp. 54 genes, including 31 protein-coding genes, 20 tRNA genes, and 3 rRNA genes, M.atropurpurea is longer than that of M. multicaulis (395,412bp),total 57 gene contain 32 protein-coding genes, 22 tRNA and 3 rRNA were annotated in the genome.

Our result indicates consistency in molecular and taxonomic classification, besides GC contents in angiosperms,. variation and evolutionary status of Morus .This study can provides extensive information about the mt genome for Morus L

Acknowledgments

We thank National Mulberry GeneBank Zhenjiang for providing the original leafs of M. multicaulis and M.atropurpurea.

Authors’ contributions

Guo Liangliang and Zhao Weiguo concieved and designed the research. GL performed, assembled the genomes, analyzed the data, and wrote the original manuscript. Shi Yishu Collected leaf samples, Wu Mengmeng extracted chloroplast DNA., Michael Ackah revised the manuscript, Guo Peng analyze portions of data, Zheng Danyan edited the manuscript, LQ editing of the final manuscript; All authors contributed to the editing of the final manuscript.

Funding

Funding was provided from five department as follows: Sericulture Industry Technology in China Agriculture Research System, Guangxi innovation driven development project, Open Program of Key Laboratory of Silkworm and Mulberry Genetic Improvement Ministry of Agriculture, China, The Crop Germplasm Resources Protection Project of the Agriculture Ministry, and National Infrastructure for Crop Germplasm Resources.

Availability of data and materials

The sequence and annotation of M. multicaulis and M.atropurpurea mt genome data was deposited in the NCBI.The accession number in Gene Banks is MW924382 and MW924383.

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Author details

1 Key Laboratory of Silkworm and Mulberry Genetic Improvement, Ministry of Agriculture, School of Biology and Technology, Jiangsu University of Science and Technology, Zhenjiang, Jiangsu, P.R. China, 2Sericultural Research Institute, Guangxi Zhuang Autonomous Regin, Nanning,530007, P.R. China

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No competing interests reported.

The Complete Mitochondrial Genome Sequence Variation and Phylogenetic Analysis of Mulberry

Status:

Version 1

Abstract

Figures

Introduction

Material And Methods

Plant material, DNA extraction, and sequencing

Assembly and annotation of the mitochondrial genome

Analysis of repeated sequences

RNA editing analyses and chloroplast to mitochondrion DNA transformation

Variation architecture and Phylogenetic tree construction

Results

Genome content and organization

Variations and Codon usage

The prediction of RNA editing

Homology analysis of chloroplast with mitochondria

Phylogenetic analysis within Dicotyledon mt genomes

Discussion

Conclusion

Declarations

References

Competing Interests

Status:

Version 1