Comparison of the Complete Chloroplast Genomes in the Astragalus

Chloroplast is an important intracellular organelle of plants for photosynthesis (Wan et al., 2009). The genome of chloroplast is circular in structure and its size varies from 120 kb to 170kb (Olmstead et al., 1994). The genome features of chloroplast are usually quadripartite configurations, including small single copy (SSC) region, large single copy (LSC) region, inverted repeats IRa and Irb (Tangphatsornruang et al., 2010). In addition Chloroplast is haploid, maternally inherited and possess high conservation in gene content, with low nucleotide substitution rate which is beneficial to study evolutionary relationship in plants at taxonomic level (Liu et al., 2019). The central rule is an important biological rule, the triplet codon transfers genetic information from mRNA to protein in the process of translation and plays an important role in the life activities of organisms (Wolfe et al., 1987). Different codons that encode the same amino acid are called synonymous codons. There are differences in the frequency of use of synonymous codons in encoding amino acids, that is, codon usage bias (Sharm et al., 1987; Archtti, 2004). For example, in monocotyledonous species such as maize, the codon often ends with G/C (Fennoy et al., 1993) and in dicotyledonous plants such as tea tree, it often ends with  A/U (Wang et al., 2018a). Natural selection, gene mutation and genetic drift are the causes of codon usage bias and the influencing factors of codon usage bias are different in different organisms (Bulmer et al., 1991). Codon usage bias is an important evolutionary feature of organisms, which can provide important information for studying biological evolution, gene function and foreign gene expression. Since the chloroplast genome is easier to sequence than the nuclear genome, the research on codon preference mainly focuses on the chloroplast genome.
       
Astragalus is the largest genus in Leguminosae and is widely distributed in the Northern hemisphere, south America and Africa. At present, more than 30 medicinal plants of Astragalus have been found, in which diterpenoids have obvious clinical effects (Lei et al., 2016). Astragalus Mongolicus and Astragalus Membranaceus are both medicinal and edible plants, used to keep healthy. The Astragalus genus is used medicinally, representing a major traditional Chinese medicinal material in our country, known for its effects of replenishing qi, consolidating the superficial resistance, detoxification, promoting pus discharge, diuresis and promoting tissue regeneration. In recent years, pharmacological research has discovered that the primary medicinal component in Astragalus is Astragaloside. Among them, Astragaloside IV is its most significant monomeric active ingredient, with effects including tumor inhibition, as well as antibacterial and anti-inflammatory properties. Although the chloroplast genomes of most plants in its family have been sequenced and annotated, there are fewer studies on comparative chloroplast genomics research and codon preference research of Astragalus. In this study, we collected three valuable medicinal Astragalus species from the northern regions and systematically compared the chloroplast genomes and codon usage characteristics of the Astragalus genus.

Plant material, DNA extraction and sequencing
 
Fresh leaf samples of Astragalus dahuricus (Pall.) DC., Astragalus melilotoides Pall.var. tenuis Ledeb.and Astragalus laxmannii Jacq. were collected in May 2022 from National Germplasm Perennial Herbage Nursery of the Inner Mongolia Horticulture Research Institute (40.57'N, 111.93'E and altitude 1040 m above sea level), Hohhot, Inner Mongolia Autonomous Region, China. These samples had been formally identified by Juan Zhang before collection, an expert in plant taxonomy. Leaf samples were frozen immediately in liquid nitrogen, conserved in drikold, extracted by the modified CTAB method (Yan et al., 2018), used for library preparation and paired-end (PE) sequencing by the Illumina Novaseq instrument at Novogene CO., Ltd (Beijing, China).
 
Genome assembly and annotation
 
Genomic DNA was extracted from fresh leaves using a Plant DNA Isolation Kit (Tiangen, Beijing, China) and sequenced using the MiSeq PE150 platform (Illumina, San Diego, CA, United States), yielding 150 bp paired-end reads, at Novogene Co. (Tianjing, China). Chloroplast genomes of Astragalus mongholicus var. dahuricus, Astragalus melilotoides var. Tenuis Ledeb. and Astragalus laxmannii Jacq. de novo assembled using NOVO Plasty (Dierckxsens et al., 2020) with default parameters. Genomes were annotated using the plastid genome annotator (PGA) tool (Biomatters Limited 2018), coupled with manually edited start and stop codons using Geneious (Pánek et al., 2022). The Astragalus mongholicus Cp genome sequence (NCBI accession number: NC029828) was used as a reference. The annotation results were checked using the Dual Organellar GenoMe Annotator (DOGMA) (Shi et al., 2019) and Cp GAVAS2 (Stephan et al., 2019). OGDRAW (version 1.3.1) (Kurtz et al., 2004) was used to draw the gene map of the Cp genomes.
 
Codon usage
 
Codons encoding the same amino acid are called synonymous codons and the difference in use frequency of synonymous codons is the CUB. In order to ensure the accuracy of the results, we eliminated sequences less than 300 bp before codon analysis (Ikemura, 1981; Wang et al., 2018b) and the CUB was calculated using Codon W v1.4.2. We also analyzed the effective number of codons (ENC) (He et al., 2016) and relative synonymous codon usage (RSCU). ENC refers to the effective number of codons and the range of its theoretical value is 20-61, representing the strength of codon bias. The larger the ENC value, the weaker the codon bias. RSCU refers to the relative probability between synonymous codons encoding corresponding amino acids for a particular codon. If there is no preference for the use of codons, the RSCU value of the codon is equal to 1.00. When the RSCU value of a codon is greater than 1.00, it indicates that the frequency of the codon use is relatively high and vice versa. The codon base composition and other relative parameters of three Astragalus were analyzed and calculated using Codon W v1.4.2 and the optimal codon usage frequency of other fungi and plants.
 
Codon preference influencing factor analysis
 
Parity rule 2 (PR2) analysis
 
We analyzed the distribution of the third base in the codon of amino acid, because it may affect the preference of codon usage (Wan et al., 2004). If codon usage is only influenced by mutations, theoretically, the frequency of using A/T and G/C at the third position of the codon should be equal. Otherwise, codon bias may be influenced by natural selection and other factors (Zhang et al., 2007). In order to analyze the composition of the third codon in CDS sequence, eight amino acids with four synonymous codons were selected for analysis, including serine (S), leucine (L), proline (P), arginine (R), threonine (T), valine (V), alanine (A) and glycine (G). The result was showed by using G₃/(G₃+C₃) and A₃/(A₃+T₃) as abscissa and ordinate respectively.
 
Neutral analysis
 
The chloroplast genomes of three Astragalus species were analyzed by neutral mapping. GC₃ content of each gene was used as abscissa, GC₁₂ as ordinate and an R package was used to draw scatter plot and make straight line fitting. If the regression coefficient is close to 1, the codon preference is mainly affected by mutations. If the regression coefficient is close to 0, the codon preference is mainly affected by natural selection (Song et al., 2017).

Chloroplast genome characteristics of Astragalus
 
The complete chloroplast genome of the three Astragalus species were found to have lost one copy of the IR region, the total lengths of the chloroplast genomes were 122,815 bp (A. dahuricus), 123,012 bp (A. melilotoides Pall. var. tenuis) and 123,729 bp (A. laxmannii Jacq.), while the total GC content ranged from 33.95% to 34.10% (Fig 1). There were 107-110 genes, including 75-76 protein-coding genes (PCGs), four ribosomal RNA genes (rRNAs) and 28-30 transfer RNA genes (tRNAs). A. dahuricus had the fewest genes and lacked atpE, trnE-UUC*, trnM-CAU (Table 1).
 

 

 
Relative synonymous codon usage analysis of the chloroplast genomes
 
The RSCU of the chloroplast genomes of three species of Astragalus was calculated using all protein-coding genes. The RSCU value is the ratio of the frequency of use of a particular codon to the expected frequency. It enables the detection of synonymous codons that do not uniformly occur in the coding sequence. Codons with no preference value are set to 1.00. The actual usage of codons with an RSCU value >1.00 is higher than expected and that of codons with an RSCU value <1.00 is lower than expected. The results showed that all species, there were 30 codons with a RSCU value greater than 1.00, of which, 29 ended with A or U codons one ended with G codon (UUG). In addition, the RSCU value of methionine (AUG) and tryptophan (UGG) was 1.00. Isoleucine was the most amino acid encoded in the chloroplast genome, accounting for 10% on average of all amino acids. Methionine had the smallest number of codons, accounting for only 1.94% on average of all amino acids (Fig 3).
 
       
Fig 2 shows the codon contents of 20 amino acids and stop codons of all protein-coding genes in the chloroplast genomes of the three species of Astragalus sequenced in this study. Among the codons exhibiting usage bias, there are 53 with a relative synonymous codon usage (RSCU) greater than 1.
 

 

 
Optimal codons
 
The ENC value of the CDS of the three Astragalus chloroplast genome is sorted from low to high and the genetic construction library of 10% of the genes at both ends are taken. Among them, the ENC value is small as a high expression library and the large value of ENC is a low expression library. The codons of the ΔRSCU> 0.08 in the two libraries are used as high expression codons and RSCU> 1.00 codons are used as high-frequency codons. The optimal codons are both high expression and high-frequency dense codons. There are 12 optimal codons that meet RSCU greater than 1 and ΔRSCU greater than 0.08. Among them, 14 best codons are shared by three Astragalus species. All the best codons ended with A/U. There are 3 ending at the end of A and 8 at the end of U (Table 2).
 

 
Determining codon preference factors
 
In this study, the parity rule 2 (PR2) of codon A/T (A₃ and T₃) and C/G (G₃ and C₃) of three Astragalus chloroplast genomes were analyzed (Fig 4). The results showed that the coordinate points were unevenly distributed in four regions, mainly distributed in G₃/(G₃+C₃) <0.5 and A₃/(A₃+T₃) <0.5. The results further showed that the usage preference of codons affected by base. In order to further determine the main factors affecting codon preference in the chloroplast genomes of Astragalus, neutral mapping analysis were conducted on the chloroplast genomes of three Astragalus. It found that GC₁₂ distributed in 0.312~0.586 and GC₃ distributed in 0.168~0.329. The regression coefficients ranged from 0.0368 to 0.0636 and GC₁₂ was positively correlated with GC₃. The results showed that the codon usage of chloroplast genome in three Astragalus plants were affected not only by natural selection, but also mutation.


 
Astragalus plants and other species of codon usage comparison
 
According to the calculations performed by Codon W, the usage frequencies of each codon in the chloroplasts of the three Astragalus species were compared with the codon usage frequencies of various organisms, including Escherichia coli, Saccharomyces, Arabidopsis thaliana, Glycine max and Nicotiana tabacum as published in the Codon Usage Database. The results indicate that when comparing with other organisms, for Astragalus plants, there is only Arabidopsis thaliana with a codon usage frequency ratio of 0.5 to 2.0. With a ratio greater than 2.0, there are 2 codons when compared with Escherichia coli and 4 codons when compared with Saccharomyces. When compared with Glycine max, there are 3 codons with a ratio greater than 2.0 and 3 codons with a ratio less than 0.5. These findings suggest that Astragalus plants are different from the use of 5 modes of plant codons usage.

Junjie Wang designed the experiment, Dabao Yin performed the experiments and wrote the manuscript, others contribute equally to the manuscript. This research received no external funding by “Integration of efficient breeding and processing of multi-functional grass seed” Award Number (2022YFDZ0025).

The authors declare that they have no conflicts of interest.