Field Muskmelon (Cucumis melo L. var. agrestis Naud.) belongs to the Cucumis of the family Cucurbitaceae, wild weeds. A number of scholars believe that the Field Muskmelon is the wild ancestor of Chinese oriental thin skin melon (Ma, 2020a). Seeds contain a lot of fat, which can be extracted from oil and eaten. Fruit rich in medicinal ingredients such as cucurbitacin, is a potential new type of wild plant (Ma, 2020b). At present, chloroplast genome sequencing fragments can be accurately assembled, but it's still difficult to assemble a larger complete genome, nuclear genome assembly is more difficult (Anton et al., 2012). Field Muskmelon is one of the most important cash crops in the world, Chloroplast genome sequencing has been successful, Field Muskmelon chloroplast genome contains 133 genes. The total GC content of the genome was 36.9% (Zhu et al., 2016). Genetic diversity of local muskmelon varieties in vietnam using chloroplast genome analysis (Phan et al., 2010). Genomic sequence polymorphisms were used to infer diversity and genetic differentiation of cultivated melon (Katsunori et al., 2013). And elaborated the melon chloroplast genome complete sequence and its systematic significance (Hong et al., 2020). So far, the study of chloroplast genome of Field Muskmelon has not been reported. In order to understand the composition of chloroplast genome and the relationship between genes and botanical traits, to provide theoretical and technical basis for breeding genetic improvement, The experiment material, Illumina high - throughput sequencing, The chloroplast genome sequencing of Field Muskmelon was completed.

1 Results and Analysis

1.1 Statistics and annotation of chloroplast genome sequencing data

Sequencing showed that the GC content of chloroplast genome was 37.45%, Q20 was 96.83% (Table 1). A total of 133 genes, tRNA37, rRNA8, mRNA88 (Table 2). Chloroplast gene function is mainly divided into photosynthetic synthesis, self-replication and other genes, there are also some genes with unknown function (Table 3).

Table 1 Sequencing data statistics

Table 2 Statistics of chloroplast gene annotation information

Table 3 Classification and statistics of chloroplast gene function

1.2 Chloroplast genome map

The chloroplast genome of Field Muskmelon is covalently closed double-stranded cyclic molecule (Figure 1), Total 156 017 bp, Includes 1 pair of reverse repeat (IR) zones (25 797 bp), 1 large single copy (LSC) area (86 336 bp) and 1 small single copy (SSR) area (18 087 bp), genome-wide GC content of 37.45%. Six ATP synthase genes and 11 NADH dehydrogenase genes.   

Figure 1 Chloroplast genome map

1.3 RSCU codon preference

Codon preference indicates that, TGG separate corresponding Trp codes (Figure 2), Met correspond to GTG, ATT, ATG and ATC codes, respectively. both Arg, Leu and Scr correspond to 6 codon codes (Figure 2). The results of codon preference analysis showed that the UAA and GCU values were higher (Table 4), while the GCG, UGC, UAG and UGA values are lower (Table 4).

Figure 2 RSCU codon preference

Table 4 RSCU codon preference

1.4 Disperse repeats

The results showed that there were more forward and palindrome repeats, but less reverse repeats and no complementary repeats (Figure 3).

Figure 3 Statistics of scattered repeated sequences

1.5 CpSSR Results

191 single nucleotide repeats in the chloroplast genome of SSR (Table 5; Table 6), 66.55 per cent; SSR 11 dinucleotides, A total of 71 dinucleotide SSR, 24.74 per cent; Four, five and six nucleotides SSR less, were 9,3 and 2, respectively (Table 5). A,T nucleotide repeats are the largest in single nucleotide repeats, fewer repeats C,G single nucleotide (Figure 4; Table 7).

Table 5 SSR Statistics of the analysis

Table 6 Results of SSR analysis

Figure 4 Quantitative statistics of various SSR types

Table 7 Result of SSR primer design

1.6 KaKs experimental results

The ratio of non-synonymous mutation rate (Ka) to synonymous mutation rate (Ks) indicates the choice. The ratio is greater than 1, which indicates that the positive selection effect is less than 1, which indicates that there is purification selection effect. KaKs value is between 0.4 and 0.8, indicating purification selection (Table 8).

Table 8 Analysis and Statistics of chloroplast gene KaKs

1.7 Analysis pi nucleic acid diversity

The higher pi values in the ssc and IR regions also mean that the region has a large degree of variation and a small value in the lsc interval, indicating a small degree of variation (Figure 5).

Figure 5 Polyline of geneo Pi value

1.8 IRscope test results

The chloroplast genome is a circular structure, IR has four boundaries with LSC and SSC, namely LSC-IRb, IRb-SSC, SSC-IRa and IRa-LSC. During the evolution of the genome, IR boundaries expand and shrink. Bringing certain genes into IR or single copy regions, Use the SVG module in the Perl to visualize the boundary information (Figure 6). There's a slight difference between several melon species, the length of the genome varies slightly, slightly different lengths between the four boundaries, only subtle differences, perhaps this subtle difference, which determines the different species of melon. The traits were different (Figure 6).

Figure 6 Changes IR chloroplast boundary

1.9 Comparison of chloroplast structures

Chloroplast structure is a ring-shaped structure. The results are similar by comparing the melon varieties of the proximal genus. Except for a few sites, the other approximations were higher (Figure 7).

Figure 7 Comparison of Chloroplast Structure

1.10 Evolution tree clustering results

The results of cluster analysis showed that common melon, melon and snake melon were one kind, while Field Muskmelon was closely related to common melon, melon and snake melon, but far from bitter gourd, pumpkin and loofah (Figure 8).

Figure 8 Evolutionary tree of chloroplast system

1.11 Homologous and collinearity analysis of chloroplast sequences

using the Mauve (http://darlinglab.org/mauve) software default parameters for genome alignment, sequence homology indicates that equine pomelo has homologous similarity with existing known melon, vietnamese melon, cucumber and other crops, except for a few loci, most of the sequences are approximate. Especially with Yue melon, common melon is more similar. far away from balsam pear and loofah (Figure 9).

Figure 9 Chloroplast sequence homology

2 Discussion and Conclusions

Referring to the genome sequencing of Field Muskmelon, we obtained the complete chloroplast sequencing and genome map of the plant. Conomon melon (156 017 bp) was found between the equinoxa and the same species, melon (Cucumis melo var. Makuwa) (156 016 bp), melon subspecies (Cucumis melo subsp. Agrestis) (156 016 bp) close. Homologous and collinear analysis of chloroplast sequences showed that equine melon was consistent with melon and common melon, but different from watermelon and pumpkin in tRNA, few genes. The results of cluster analysis showed that the Field Muskmelon was closest to the common melon, and the melon was closest to the common melon, snake melon and Yue melon. The number of introns in Field Muskmelon was 21, which was basically consistent with that in common melon (Wang, 2017). Among the cucurbitaceae plants, the Field Muskmelon and common melon have the same origin, which is consistent with the previous research that the Field Muskmelon is the wild ancestor of Chinese thin skin melon (Ma, 2020a). The chloroplast genome is relatively conserved and can better explain the evolution and kinship of species. and this study shows that the genetic and taxonomic results of the Field Muskmelon are consistent with those of previous studies (Ma, 2020a). Melon has a GC content of 36.9. Cucumber is also 36.9%(Zhu et al., 2016), and the GC content of Field Muskmelon is 37.45%. and the LSC content is 55.34%; SSC 11.59%, The IRs content of 33.1% was basically the same as that of melon (Zhu et al., 2016).

3 Materials and Methods

3.1 Plant materials

Fresh leaves of the Field Muskmelon plant. The collection site is Fuyang Academy of Agricultural Sciences Science and Technology Park.

3.2 Bioinformatics analysis

The Clean Data were assembled according to the chloroplast genome sequence of the reference species, the results of chloroplast sequence assembly were obtained, the results of chloroplast sequence assembly were annotated with gene structure, the chloroplast genome map was made, and the basic contents such as chloroplast genome SSR were analyzed. KaKs analysis, phylogenetic analysis and other advanced analysis content. Use fastp (version 0.20.0, https://github.com/OpenGene/fastp) software to filter raw data. The filtering criteria are as follows:

(1) Sequencing connectors and primer sequences in the Reads were removed

(2) Filter out reads whose average mass is less than Q5

(3) Filter out reads with N number greater than 5

Using OGDRAW (https://chlorobox.mpimp-golm.mpg.de/OGDraw.html) to make chloroplast genome maps. Because of the characteristics of codon biology, the codon usage rate of different species is very different. This inequality in the use of synonymous codons is called codon preference (Relative Synonymous Codon Usage, RSCU). This preference is considered to be a combination of natural selection, species mutation and genetic drift. The calculation methods are as follows: (the number of codon encoding one of the amino acids / the number of all codon encoding the amino acid) / (1/the type of codon encoding the amino acid), and (the actual frequency of codon / the theoretical frequency of the codon). Calculate using a self-written Perl script.

Scattered in repeated sequences using vmatch v2.3.0 (http://www.vmatch.de/) software combined with Perl scripts to identify repeat sequences. the parameters are set as: minimum length (minimum length)=30 bp, hamming distance (hamming distance), and the identification forms are four: forward (Forward), palindrome (Palindromic), reverse (Reverse), complementary (Complement).

SSR markers on the chloroplast genome are called cpSSR markers. Using MISA v1.0 software to carry out cpSSR analysis, the parameters 1-8 (single base repeat 8 times and above) were 2-5,3-3,4-3,5-3,6-3.

Pi (nucleic acid diversity) can reveal the variation of nucleic acid sequences of different species, and regions with high variation can provide potential molecular markers for population genetics. The homologous gene sequences of different species were compared globally by mafft software (auto mode), and the pi values of each gene were calculated by dnasp5.

Using software CGVIEW (http://stothard.afns.ualberta.ca/cgview_server/) default parameters, comparative analysis of chloroplast genome structure was performed for near-source species.

The whole genome is used for evolutionary tree analysis by default, the ring sequence is set the same starting point, and the interspecies sequence is compared by MAFFT software (v7.427,--auto mode). The compared data are trimmed with trimAl (v1.4.rev15), then the RAxML v8.2.10(https://cme.h-its.org/exelixis/software.html) software is used to select GTRGAMMA model, rapid Bootstrap analysis, bootstrap=1 The maximum likelihood evolution tree is constructed.

3.3 Genome assembly

To reduce the complexity of the next sequence assembly, bowtie2 (v2.2.4, http://bowtie-bio.sourceforge.net/bowtie2/index.shtml) was used very-sensitive-local align the database and sequence. The assembly core module uses SPAdes [1]v3.10.1(http://cab.spbu.ru/software/spades/) software to assemble chloroplast genome. Three aspects of genome quality control are performed to ensure the accuracy of assembly results:

(1) reads specific genomes, statistics of genome coverage, size of inserted fragments, etc;

(2) Genome alignment reference sequences to see collinear analysis of genome conservation and rearrangement.

(3) Genome alignment of reference sequence structure information to compare the differences between the two.

A reference sequence Cucumis_melo: MT240857.1. is used for this project.

Author’s contributions

Ma Zongxin is responsible for project design, data processing and gene sequencing, and completes the writing and revision of the paper. I have read and approved the final manuscript.

Acknowledgements

This research is supported by key R & D projects (1704 f0704067) of Anhui Science and Technology Department.

References

Anton B., Sergey N., and Dmitry A., 2012, A new genome assembly algorithm and its applications to single-cell sequencing, J Comput Biol.,19(5):455-477

https://doi.org/10.1089/cmb.2012.0021

PMid:22506599 PMCid:PMC3342519

Hong C., Wei P.K., Zhang M.M., and Hou D., 2020, The complete chloroplast genome of Cucumis melo L.'Shengkaihua'(Cucurbitaceae) and its physiognomic implications, Mitochondrial DNA Part B, 5(2):1253-1254

https://doi.org/10.1080/23802359.2020.1731364

Katsunori T., Yukari A., Kenji F., Tatsuya Y., Yasheng A., Hidetaka N., Chun L.L., Hiromichi Y., YoIchiro S., and Kenji K., 2013, Diversification and genetic differentiation of cultivated melon inferred from sequence polymorphism in the chloroplast genome, Breeding Science, 63(2):183-196

https://doi.org/10.1270/jsbbs.63.183
PMid:23853513 PMCid:PMC3688380

Ma Z.X., 2020a, Brief history of production of Cucumis melo L. var. agrestis Naud., Nongcun Jingji Yu Keji (Rural Economy and Science-Technology), 31(7):76-77

Ma Z.X., 2020b, Investigation on new food resources of a new wild plant, Nongye Yu Jishu (Agriculture and Technology), 40(1):32-34

Phan T.P.N., Yukari A., Tran T.M.H., Katsunori T., Yasheng A., Tatsuya Y., Hidetaka N., Long C.L., and Kenji K., 2010, Genetic diversity in Vietnamese melon landraces revealed by the analyses of morphological traits and nuclear and cytoplasmic molecular markers, Breeding Science, 60(3):255-266

https://doi.org/10.1270/jsbbs.60.255

Wang H.G., Bai YL., Xu H.J., Liu S.H., Zhang W.J., Liu Q., Zhang X.M., Qiao M.Q., and Wang Y., 2017, Construction and stability of cucumber chloroplast expression vector, Nankai Daxue Xuebao (Acta Scientificarum Naturalium Universitatis Nankaiensis), 50(5):61-66

Zhu Q.L., Gao P., Liu S., Amanullah S., and Luan F.S., 2016, Comparative analysis of single nucleotide polymorphisms in the nuclear, chloroplast, and mitochondrial genomes in identification of phylogenetic association among seven melon (Cucumis melo L.) culturals., 66(5):711-719

https://doi.org/10.1270/jsbbs.16066
PMid:28163587 PMCid:PMC5282756