Analysis of the chloroplast genome sequence characteristics and its code usage bias of Citrus maxima (L.) Osbeck ‘Majiayou’

Abstract：【Objective】Citrus maxima (L.) Osbeck ‘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 ‘Majiayou’industry. It is urgent to trace the origin of C. maxima (L.) Osbeck ‘Majiayou’to ensure its authenticity. There have been some studies indicating that the genetic relationship between C. maxima (L.) Osbeck ‘Maji-ayou’ and C. maxima (L.) Osbeck ‘Xinmuyou’in the surrounding areas is relatively close, and it is quite likely that C. maxima (L.) Osbeck‘Majiayou’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‘Majiayou’. The study aimed to rectify the source of C. maxima (L.) Osbeck ‘Majiayou’and explore the phylogenetic relationship with other Citrus plants through surveying the characteristics of the chloroplast genome of C. maxima (L.) Osbeck ‘Majiayou’ and its codon preference.【Methods】The total DNA extraction from the leaves of C. maxima (L.) Osbeck ‘Majiayou’was performed using an improved CTAB method. The purity of the DNA was detected using the NanoDrop 2000 spectrophotometer method; Preliminary quantification of the DNA library using Invitrogen Qubit® 2.0 fluorescence quantitative instrument method; The detection of inserted fragments in the DNA library was carried out using the Agilent 2100 biological analyzer system; The accurate quantification of the effective concentration in the DNA library was carried out using real-time fluorescence quantitative PCR method; The DNA library was sequenced using the DNBSEQ-T7 sequencer method. The assembly of the chloroplast genome was carried out using Noveplastys and CAP3 software; The annotation of the chloroplast genome was performed using GeSeq and tRNAscan- SE software; The production of the chloroplast genome map 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 were conducted using CGViewServer software; The SSR analysis of the chloroplast genome was performed using MISA software; The Longrepeat analysis of the chloroplast genome was performed using REPuter software; The calculation and analysis of the RSCU of the chloroplast genome were carried out using CodonW software; The drawing of chloroplast genome variation circles and the calculation of sequence similarity for C. maxima (L.) Osbeck ‘Majiayou’and its 18 congeneric species were performed using Gview software; The mapping of IR structural variations in chloroplast genomes of C. maxima (L.) Osbeck ‘Majiayou’and its 18 congeneric species was performed using IRscope software; The calculation of the chloroplast genome Pi of C. maxima (L.) Osbeck ‘Majiayou’and its 18 congeneric species was carried out using NADnaSP6.0 software; The sequence alignment and tree construction of chloroplast genomes of C. maxima (L.) Osbeck‘Majiayou’and its 85 same family species, as well as three outer groups of Glycosmis, were carried out using MAFFT 7.0 software and FastTree 2.1.10 software, respectively.【Results】The chloroplast genome of C. maxima (L.) Osbeck ‘Majiayou’had a total length of 160 186 bp, including 1 LSC region (87 791 bp), 1 SSC region (18 395 bp), and 2 IR regions (including IRa and IRb, both 27 000 bp). Its structure presented a typical closed circular double stranded structure. The average GC content of the chloroplast genome of C. maxima (L.) Osbeck ‘Majiayou’was 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 ‘Majiayou’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 ‘Majiayou’, including only single nucleotide repeat sequences and trinucleotide repeat sequences. The single nucleotide repeat sequences were mostly A and T repeats. A total of 34 long repeat sequences were detected in the chloroplast genome of C. maxima (L.) Osbeck ‘Majiayou’, including 13 dispersed repeat D (1739- 135 819 bp) and 21 palindrome repeat P (421-125 236 bp). The chloroplast genome sequences of C. maxima (L.) Osbeck ‘Majiayou’and its 18 congeneric 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 ranges of the nucleotide diversity in the chloroplast genome of C. maxima (L.) Osbeck‘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‘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‘Majiayou’and its 18 congeneric 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‘Majiayou’and its 18 related species was GC3＜GC2＜GC1, with an ENC value ranging from 26.309 to 61 and an average of 48.04. The codon bias was weak, and all codons except UGG, UUG, and AUG ended in A and U. Neutral plot analysis showed that the GC3 and GC12 contents of the chloroplast genes of C. maxima (L.) Osbeck ‘Majiayou’and its 18 congeneric 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‘Majiayou’and its 18 congeneric species was mainly influenced by the natural selection, and was less affected by internal mutation pressure. The 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‘Majiayou’and its 18 congeneric 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. The PR2 plot analysis showed that the chloroplast genomes of C. maxima (L.) Osbeck‘Majiayou’and its 18 congeneric 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 ‘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‘Majiayou’, including AAU, UGU, AAA, UUU, GCU, GGA, CCA, ACU, CGU, and AGU, all ending in A and U. C. maxima (L.) Osbeck‘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), and C. maxima (Burm.) Merr.‘Guanximiyou’ (MN782007, source: Fujian).【Conclusion】C. maxima (L.) Osbeck ‘Majiayou’is a relatively unique variety in the Citrus genus. The research results would provide a theoretical basis for further research on the genetic resources, species identification, and phylogenetic analysis of C. maxima (L.) Osbeck‘Majiayou’.