Analysis of the Chloroplast Genome Sequence Characteristics and its Code Usage Bias of Citrus maxima (L.) Osbeck cv. ‘Majiayou’

Abstract：【Objective】Citrus maxima (L.) Osbeck cv. ‘Majiayou’ was approved by the former Ministry of Agriculture as a national geographical indication agricultural product in 2010. At present, all counties and cities in Shangrao City are vigorously developing the C. maxima (L.) Osbeck cv. ‘Majiayou’ industry. It is urgent to trace the origin of C. maxima (L.) Osbeck cv. ‘Majiayou’ to ensure its authenticity. There are studies indicating that the genetic relationship between C. maxima (L.) Osbeck cv. ‘Majiayou’ and C. maxima (L.) Osbeck cv. ‘Xinmuyou’ in the surrounding areas is relatively close, and it is highly likely that it is a variant strain derived from the bidirectional (natural and artificial) selection of local pomelo. However, the above research has not yet solved the phylogenetic problem of C. maxima (L.) Osbeck cv. ‘Majiayou’. In order to achieve the goal of rectifying the source of C. maxima  (L.) Osbeck cv. ‘Majiayou’, exploring the phylogenetic relationship between it and Citrus plants is beneficial for molecular identification of it and its counterfeit products. At present, research on Pomelo mainly focuses on bagging treatment, nutritional components, photosynthetic characteristics, pollen sensitivity, pollinating insects, fermentation technology, anatomical observation, soil nutrients, etc. However, there have been no reports on the chloroplast genome characteristics and codon preference analysis of C. maxima (L.) Osbeck cv. ‘Majiayou’. Its evolutionary origin and phylogenetic relationships have not been clearly identified. In order to clarify the structural characteristics of the chloroplast genome of C. maxima (L.) Osbeck cv. ‘Majiayou’ and its phylogenetic relationship with related taxa, elucidate its taxonomic position in the Citrus genus, the characteristics of the chloroplast genome of C. maxima (L.) Osbeck cv. ‘Majiayou’ and its codon preference were analyzed in this study. 【Method】The total DNA extraction from leaves of C. maxima (L.) Osbeck cv. ‘Majiayou’ was performed using an improved CTAB method. The purity of DNA in the leaves of C. maxima (L.) Osbeck cv. ‘Majiayou’ was detected using the NanoDrop 2000 spectrophotometer method; Preliminary quantification of DNA library from leaves of C. maxima (L.) Osbeck cv. ‘Majiayou’ using Invitrogen Qubit ® 2.0 fluorescence quantitative instrument method; The detection of inserted fragments in the DNA library of C. maxima (L.) Osbeck cv. ‘Majiayou’ leaves was carried out using the Agilent 2100 biological analyzer system; The accurate quantification of the effective concentration in the DNA library of C. maxima (L.) Osbeck cv. ‘Majiayou’ leaves was carried out using real-time fluorescence quantitative PCR method; The DNA library of C. maxima (L.) Osbeck cv. ‘Majiayou’ leaves was sequenced using the DNBSEQ-T7 sequencer method. The assembly of the chloroplast genome of C. maxima (L.) Osbeck cv. ‘Majiayou’ was carried out using Noveplastys and CAP3 software; The annotation of the chloroplast genome of C. maxima (L.) Osbeck cv. ‘Majiayou’ was performed using GeSeq and tRNAscan-SE software; The production of the chloroplast genome map of C. maxima (L.) Osbeck cv. ‘Majiayou’ was carried out using OGDRAW software. The analysis and statistics of GC content in the large single copy region (LSC), small single copy region (SSC), and reverse repeat region (IR) of the chloroplast genome of C. maxima (L.) Osbeck cv. ‘Majiayou’ were conducted using CGViewServer software; The SSR analysis of the chloroplast genome of C. maxima (L.) Osbeck cv. ‘Majiayou’ was performed using MISA software; The Longrepeat analysis of the chloroplast genome of C. maxima (L.) Osbeck cv. ‘Majiayou’ was performed using REPuter software; The calculation and analysis of the RSCU of the chloroplast genome of C. maxima (L.) Osbeck cv. ‘Majiayou’ were carried out using CodonW software; The drawing of chloroplast genome variation circles and the calculation of sequence similarity for C. maxima (L.) Osbeck cv. ‘Majiayou’ and its 18congeneric species were performed using Gview software; The mapping of IR structural variations in chloroplast genomes of C. maxima (L.) Osbeck cv. ‘Majiayou’ and its 18congeneric species was performed using IRscope software; The calculation of the chloroplast genome Pi of C. maxima (L.) Osbeck cv. ‘Majiayou’ and its 18congeneric species was carried out using NADnaSP6.0 software; The sequence alignment and tree construction of chloroplast genomes of C. maxima (L.) Osbeck cv. ‘Majiayou’ and its 44congeneric species, as well as two outer groups of Glycosmis, were carried out using MAFFT 7.0 software and FastTree 2.1.10 software, respectively. 【Result】The chloroplast genome of C. maxima (L.) Osbeck cv. ‘Majiayou’ had a total length of 160186 bp, including 1 LSC region (87791 bp), 1 SSC region (18395 bp), and 2 IR regions (including IRa and IRb, both 27000 bp). Its structure presents a typical closed circular double stranded structure. The average GC content of the chloroplast genome of C. maxima (L.) Osbeck cv. ‘Majiayou’ is 38.47%, with the GC content in the IR region being higher than that in the LSC and SSC regions. The chloroplast genome of C. maxima (L.) Osbeck cv. ‘Majiayou’ has annotated 133 functional genes, including 88 coding sequence (CDS) genes, 8 ribosomal RNA (rRNA) genes, and 37 transporter RNA (tRNA) genes. A total of 79 simple repeat sequences (SSRs) were detected in the chloroplast genome of C. maxima (L.) Osbeck cv. ‘Majiayou’, including only single nucleotide repeat sequences and trinucleotide repeat sequences. The single nucleotide repeat sequences are mostly A and T repeats. A total of 34 long repeat sequences were detected in the chloroplast genome of C. maxima (L.) Osbeck cv. ‘Majiayou’, including 13 dispersed repeat D (1739-135819 bp) and 21 palindrome repeat P (421-125236 bp). The chloroplast genome sequences of C. maxima (L.) Osbeck cv. ‘Majiayou’ and its 18congeneric species were highly conserved, with significant sequence differences between genes such as petN, petL, psbI, psbK, psaI, pafII, trnT-GGU, trnR-UCU, trns-GGA, and trnL-UAA in the LSC and SSC regions. The variation range of nucleotide diversity in the chloroplast genome of C. maxima (L.) Osbeck cv. ‘Majiayou’ was from 0 to 0.00629; The degree of variation in the non coding region of the chloroplast genome of C. maxima (L.) Osbeck cv. ‘Majiayou’ was higher than that in the gene coding region. The overall variability was higher in the LSC region, followed by the SSC region. The IR region had the lowest variability and was the most conservative region; The SC/IR boundaries of the chloroplast genomes of C. maxima (L.) Osbeck cv. ‘Majiayou’ and its 18congeneric species were relatively conservative. The bias analysis of synonymous codons showed that the variation trend of GC content at three positions of the chloroplast genome codon of C. maxima (L.) Osbeck cv. ‘Majiayou’ and its 18 related species was: GC3C. maxima (L.) Osbeck cv. ‘Majiayou’ and its 18congeneric species were mostly distributed above the diagonal, with an internal mutation contribution rate of only 2.5% and a natural selection contribution rate of 97.5%. The codon usage bias of the chloroplast genome of C. maxima (L.) Osbeck cv. ‘Majiayou’ and its 18congeneric species was mainly influenced by natural selection, and was less affected by internal mutation pressure. ENC plot analysis showed that there were significant differences between the actual and expected values of most of the genes ENC in the chloroplast genome of C. maxima (L.) Osbeck cv. ‘Majiayou’ and its 18congeneric species, and the distribution of GC3 values was relatively concentrated, indicating that natural selection was an important factor affecting the codon usage bias of chloroplast genome of C. maxima (L.) Osbeck cv. ‘Majiayou’ and its 18congeneric species. PR2 plot analysis showed that the chloroplast genomes of C. maxima (L.) Osbeck cv. ‘Majiayou’ and its 18congeneric species exhibited C>G and T>A phenomena at the third synonymous codon position, indicating that the codon usage preference of C. maxima (L.) Osbeck cv. ‘Majiayou’ was influenced not only by internal mutations but also by natural selection. There were a total of 10 optimal codons in the chloroplast genome of C. maxima (L.) Osbeck cv. ‘Majiayou’, including AAU, UGU, AAA, UUU, GCU, GGA, CCA, ACU, CGU, and AGU, all ending in A and U. C. maxima (L.) Osbeck cv. ‘Majiayou’ was closely related to C. maxima (‘Dongshizaoyou’ in Xishuangbanna, KY055833, source: Yunnan), Japanese summer orange (C. natsudaidai, ON193075, source: South Korea), C. maxima ‘Liuyuezao’ (MT527726, source: Fujian), C. maxima (Burm.) Merr. ‘Guanximiyou’ (MN782007, source: Fujian). 【Conclusion】 C. maxima (L.) Osbeck cv. ‘Majiayou’ is a relatively unique variety in the Citrus genus. The research results provide a theoretical basis for further research on the genetic resources, species identification, and phylogenetic analysis of C. maxima (L.) Osbeck cv. ‘Majiayou’.

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