The Complete Mitochondrial Genome Sequence Variation and Phylogenetic Analysis of Mulberry

Mulberry is admired for its landscaping and possesses high development prospects and scientic 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 reects 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. all a better understanding of the genetics of the Morus L. 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 classication. 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. 2016).So identication 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. tRNA genes mitochondria. the cytoplasmic genome, migration of cpDNA to the mt genome 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 Transfer of tRNA genes from cp to mt in angiosperms(Bi 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 signicant differences with other terrestrial green plants.nucleotide diversity (pi) of the mt genome in M. multicaulis calculated.In our research found 10 gene ranged from 0.00063 to 0.02182 maybe relative to we eudicot complete 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 classication, besides GC contents in angiosperms,. variation and evolutionary status of Morus .This study can provides extensive information about the mt genome for Morus L


Introduction
Morus is an economically signi cant 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 signi cant differences in length,gene sequence and content ( & 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 con gurations (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 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 nal 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 con rmed the nal annotation result is obtained.Use OGDRAW (https://chlorobox.mpimp-golm.mpg.de/OGDraw.html) to drawn a circular mitochondrial genome map.
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).   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-ve 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. 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 shu e-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-speci c and each group shows nearly identical patterns among themselves.The vista plot patterns produced are remarkably group-speci c 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 identi ed 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. 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 classi cation. 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).

Discussion
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 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 signi cant 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).

Conclusion
In this study, we collected two cultivated species The circular map of the mt genome of M.multicaulis Figure 2 Page 16/21 The circular map of of the mt genome of M.atropurpurea. The forward direction gene encoding is located outside the circle, and reverse direction is located inside. The inner gray circle represents the GC content.     The distribution of RNA-editing sites in M.multicaulis mt genome protein-coding  Percent identity plot for comparison of three Morus L relative to Eudiots.

Figure 10
Page 20/21 The nucleotide diversity (pi) of of M.multicaulis.mt genome Figure 11 Phylogenetic analysis of Morus species using the complete mt genome by the ML method.

Figure 12
Phylogenetic analysis of Morus species using the complete mt genome by the NJ method.