Characteristics of Complete Chloroplast Genome of Grevillea robusta

doi:10.21203/rs.3.rs-3250429/v1

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Characteristics of Complete Chloroplast Genome of Grevillea robusta

https://doi.org/10.21203/rs.3.rs-3250429/v1

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Grevillea robusta is an important plant in Proteaceae, and decoding and understanding the chloroplast genome of Grevillea robusta is of great theoretical significance and practical value to the genetic diversity and phylogenetic relationship of Proteaceae. On the basis of high-throughput sequencing data of Grevillea robusta, we assembled and annotated the sequencing results using GetOrganelle and CPGAVAS2 programs, and downloaded the chloroplast genome data of genera Macadamia, Helicia and Protea from NCBI database. The chloroplast genomes of four generas. The length of chloroplast genome of Grevillea robusta was 158,642 bp, consisting of 129 genes, including 84 protein-coding genes, 37 tRNA genes and 8 rRNA genes. 56 SSRs were obtained from Grevillea robusta, among which the single nucleotide repeats were the most (66.07%) and the six nucleotide repeats were the least (1). At the same time, 34 repeats were detected in chloroplast genome of Grevillea robusta, mainly are palindrome repeats (16). The IR region of Grevillea robusta didn’t experience a significant contraction/expansion event, whereas Protea kilimandscharica showed a dramatic contraction. Gene selection pressure analysis showed that ycf1 genes showed positive selection signals. Analysis of RNA editing sites showed that there were 148 RNA editing sites in the protein-coding genes of chloroplast genome of Grevillea robusta, and most of them are C/U editing, up to 54.73%. Phylogenetic analysis confirmed that Grevillea robusta was belongs to Proteaceae, and grouped with Helicia and Macadamia, with a support rate of 100%. The chloroplast genome of Grevillea robusta was assembled successfully, which had high similarity with the chloroplast genome of Helicia and Macadamia, and was clustered into a branch during the phylogeny of Proteaceae. The results of this study laid a foundation for understanding the systematic evolution of the Proteaceae plants, and provide rich data supported for the development of molecular biological information such as molecular marker.

Proteaceae

high-throughput sequencing

phylogenetic relationship

Protea kilimandscharica

Grevillea robusta A. Cunn. ex R. Br. is a tree of the genus Grevillea in the family Proteaceae, native to Australia, with beautiful shape, bright flowers, peculiar leaves and strong absorption ability of harmful gases, which is an ideal tree species for urban greening (Weng QJ et al. 2006). At the same time, Grevillea robusta has the characteristics of drought and infertile tolerance and strong adaptability, which is important for the comprehensive management of stone desertification mountain (Gong Zheng et al. 2013). In recent years, domestic and foreign scholars have focused on the analysis of extract components and activity assay of Grevillea robusta, for example, some studies found that leaf extracts of Grevillea robusta showed toxicity against grain storage pests and termites (Bhuvaneswari et al., 2014; Afzal et al. 2019), in addition, the phenolic substances in Grevillea robusta showed some anticancer activity and antimalarial In addition, phenolic substances in Grevillea robusta showed some anticancer and antimalarial activities (Ahmed et al. 2000; Chuang and Wu 2007). However, the genetic analysis and molecular phylogeny of Grevillea robusta have been less studied, and only one study has developed five SSR markers for it based on illumina low-depth high-throughput technology (Dabral et al. 2021), and no chloroplast genomic information has been available for Grevillea robusta, which may limit our knowledge of this species.

Chloroplasts are important organelles of plants and a highly conserved tissue, whose genomic inheritance is mainly uniparental and the recombination rate of the evolutionary process is low, which can better reflect the evolutionary and developmental process of the population. In addition, the fast evolutionary rate of protein noncoding sequences in the chloroplast genome can provide some meaningful information loci that are important for the identification of plant species (Petit et al. 2005). Therefore, the chloroplast genome (cpDNA) is considered as an ideal barcode for studying plant phylogeny. In angiosperms, most chloroplast genomes are a double-stranded circular tetrad structure consisting of a large copy region (LSC), a small copy region (SSC), and two inverted repeat sequences (IRa/IRb) (Jansen and Ruhlman 2012). The chloroplast genome sequence ranges mostly within 150–170 kb (Terakami et al. 2012) and contains roughly 110–130 tRNA genes, rRNA genes, and protein-coding genes involved in photosynthesis (Choi et al. 2015). Currently, information from the chloroplast genome has been used in various studies, such as the development of DNA barcodes, chloroplast transformation and breeding of crops with superior agronomic traits (Bock and Khan 2004; Daniell et al. 2005)

In this paper, we sequenced, assembled and analyzed the chloroplast genomes of Grevillea robusta based on high-throughput sequencing (NGS), and compared them with those of Helicia shweliensis W. W. Sm., Macadamia integrifolia Maiden & Betche and Protea kilimandscharica Engl. from Proteaceae, with the aim of enriching the genomic resources of Grevillea robusta and providing information for the study of plant evolutionary patterns and phylogeny in the Proteaceae.

1.1 Test materials

Fresh Grevillea robusta leaves were collected from the campus of Southwest University, Beibei District, Chongqing (geospatial coordinates: N29.842889,E106.394527). Samples were preserved in the herbarium of Southwest University with voucher number YJ-066. Total genomic DNA was extracted by CTAB method (Arseneau et al. 2017) and total DNA fragments were detected using ultrasound. DNA libraries with an insert size of 350 bp were constructed with the help of the NEBNext (Emerman et al. 2017) library construction kit and sequenced using the HiSeq Xten PE150 sequencing platform. The sequencing yielded a total of 4.57 Gb of raw data, containing 15,249,412 reads, and clean data were obtained by Trimmomatic (Bolger et al. 2014): we removed more than 5% of low quality sequences with base N and more than 50% of base quality values Q < 19. Finally, 15,158. 092 clean reads were obtained after trimming.

1.2Assembly and annotation of genes

Chloroplast assembly was done using GetOrganelle (v1.7.3) to assemble the chloroplast genome with default parameters based on clean reads. The original sequencing data were mapped to the assembled chloroplast genome using Bowite2 (v2.96 0.1) (Langmead et al. 2009) under default settings to verify the accuracy of the assembly. Based on the assembly results annotation of the Grevillea robusta chloroplast genome was completed with the help of the CPGAVAS2 (Tillich et al. 2017) program and the reference genome (Macadamia ternifolia, GenBank: NC_036416.1), and annotation errors were manually edited and corrected using Appolo software. The genome sequence has been saved in GenBank with the accession number OK054586.1.

1.3 Repeated sequences and SSR analysis

Simple repeat sequences (SSRs) were obtained through the online website MISA (https://webblast.ipk-gatersleben.de/misa/) and include single, two, three, four, five and six nucleotide repeats with minimum numbers of 10, 5, 4, 3, 3 and 3, respectively. REPuter (https://bibiserv.cebitec.uni-bielefeld.de/reputer) was used for the calculation of palindromic repeats, reverse repeats, and dispersed repeats, etc. The parameters were set as follows:Hamming distance of 3, the minimum repeat size is 30 bp, and the e-value is limited to less than 1e-05.

1.4 Sequence diversity analysis

We used the online website IRscop (https://irscope.shinyapps.io/irapp/) to visualize the chloroplast genome IR/SC boundary and its adjacent genes. Shared protein-coding gene sequences were extracted in Phylosuite software based on annotation files in .gb format, and sequences were aligned using Mafft software. The CODEML model in PAML v4.9 (Yang 1997) was used to calculate the nucleotide substitution rate of protein-coding genes for each chloroplast, including the non-synonymous substitution rate (Ka), the synonymous substitution rate (Ks) and the ratio of both (Ka / Ks). The detailed parameters are as follows: verbose = 0; icode = 0; weighting = 0; commonf3x4 = 0 ; ndata = 1. For each protein-coding gene, we constructed a single-gene tree as a background tree using the maximum likelihood (ML) method implemented in RAxML v8.2.4.

1.5 Identification of RNA editing sites

We downloaded the Grevillea robusta transcriptome data from NCBI's SRA database (accession number: ERR2040150) and mapped the raw transcriptome data to the protein-coding gene sequences of Grevillea robusta chloroplasts, using our custom Python script to count the base coverage and specific base composition of each locus to identify loci where RNA editing events had occurred. To exclude false positive loci triggered by natural variation, we also downloaded another DNA-seq data set for Grevillea robusta (SRR11309581) and mapped all reads from this data set to the nucleotide sequences of our assembled Grevillea robusta chloroplast protein-coding genes to identify potential single nucleotide polymorphism loci (SNPs). Among the RNA editing sites identified by the transcriptome, we only retained sites with coverage greater than 5 and carefully screened for reads that may have been mapped up incorrectly due to sequence homology, and finally excluded previously identified SNP sites, and the remaining sites will be retained as high-quality RNA editing sites.

1.6 Construction of phylogenetic tree

Download from NCBI repository Helicia shweliensis NC_045942.1, Helicia nilagirica NC_057271.1, Macadamia integrifolia KF862711.1, Macadamia ternifolia NC_036416.1, Protea kilimandscharica, Platanus occidentalis DQ923116.1, Platanus racemosa MZ128519.1, Sabia parviflora MW566751.1, Meliosma aff. cuneifolia NC_029430.1, Nelumbo lutea JQ336992.1, Nelumbo nucifera JQ336993.1, Coptis chinensis NC_036485.1 and Coptis teeta NC_054331.1 chloroplast genomes for phylogenetic analysis.

The chloroplast genomes of the 16 species were multiplexed and saved as .phy format files. Coptis chinensis and Coptis teeta were selected as outgroups, and maximum likelihood trees were constructed using IQtree v2.0 with the command "iqtree2 -s sequences.phy --alrt 1000 -B 1000 ", and finally embellish the tree results with ITOL.

1.1 Chloroplast genome characteristics of Grevillea robusta

The chloroplast genome of Grevillea robusta is a typical tetrameric ring structure (Fig. 1), with a total genome sequence length of 158,642 bp, and a GC content of 38.13%, of which the LSC, SSC and IR regions were 87,021 bp, 18,961 bp and 26,330 bp, respectively .The GC content of the IR region (43.24%) was significantly higher than that of the SSC region ( The GC content of the IR region (43.24%) was significantly higher than that of the SSC region (31.78%) and the LSC region (36.43%). The Grevillea robusta chloroplast genome contained 129 genes, including 37 tRNAs, 8 rRNAs and 84 protein-coding genes. Grevillea robusta did not differ significantly from the other three genus species of the Proteaceae in terms of genome length, gene content and total GC content (Table 1). Grevillea robusta contained 16 intron-containing genes, clpP and ycf3 genes had two introns, and 14 genes contained one intron (Table 2).

Table 1

Comparison of chloroplast genome characteristics of four genera in *Proteaceae*
Species		G.robusta	H.shweliensis	M. integrifolia	P.kilimandscharica
Accession number		OK054586.1	NC_045942.1	KF862711.1	MH362765.1
Length (bp)	Total	158,642	157,151	159,714	158,657
	LSC	87,021	85,490	88,092	87,241
	SSC	18,961	18,256	18,812	18,534
	IR	26,330	26,701	26,405	26,441
GC content (%)	Total	38.13	38.11	38.12	38.03
	LSC	36.43	36.46	36.54	36.31
	SSC	31.76	31.77	31.67	31.56
	IR	43.24	42.93	43.03	43.14
Gene numbers	Total	129	129	129	128
	tRNA	37	36	36	36
	rRNA	8	8	8	8
	Protein-coding	84	85	85	84

Table 2

Gene composition in the chloroplast genome of *Grevillea robusta*
Category of Genes	Group of Genes	Name of Genes
Ribosomal RNA	rRNA	rrn16S(x2),rrn23S(x2),rrn5S(x2),rrn4.5S(x2)
Transfer RNA	tRNA	37 unique trna genes
Photosynthesis	Subunits of ATP synthase	atpA, atpB, atpE, atpF, atpH, atp*I
	Subunits of photosystem II	psbA, psbB, psbC, psbD, psbE, psbF, psbI, psbJ, psbK, psbM, psbN, psbT, psbZ, ycf3**
	Subunits of NADH-dehydrogenase	ndhA, ndhB(x2), ndhC, ndhD, ndhE, ndhF, ndhG, ndhH, ndhI, ndhJ, ndhK
	Subunits of cytochrome b/f complex	petA, petB, petD, petG, pet*N
	Subunits of photosystem I	psaA, psaB, psaC, psaJ
	Subunit of rubisco	rbcL
Self-replication	Large subunit of ribosome	rpl14, rpl16, rpl2(x2), rpl20, rpl22, rpl23(x2), rpl32, rpl33, rpl*36
	DNA dependent RNA polymerase	rpoA, rpoB, rpoC1, rpo*C2
	Small subunit of ribosome	rps11, rps12(x2), rps14, rps15, rps16, rps18, rps19, rps2, rps3, rps4, rps7(x2), rps*8
Other genes	Subunit of Acetyl-CoA-carboxylase	accD
	c-type cytochrom synthesis gene	ccsA
	Envelop membrane protein	cemA
	Protease	clpP^**
	Translational initiation factor	infA
	Maturase	matK
Unknown	Conserves open reading frames	ycf1, ycf15(x2), ycf2 (x2), ycf*4
Note: (×2) indicates that the gene is located in IRs and therefore has two complete copies, * and ** indicate that the gene contains 1 / 2 introns.

1.2 Replication and SSR analysis

Microsatellites, also known as simple repeat sequences (SSRs), are usually 6 bp tandem sequences in the genomes of eukaryotic organisms (Lovin et al. 2009), and the high polymorphism and co-dominant inheritance of SSRs have led to a wide range of applications in different types of molecular markers (Morgante et al. 2002). There are 34 oligonucleotide repeats in the chloroplast genome of Grevillea robusta, of which palindromic repeat sequences are the most abundant with 16, followed by dispersed repeats and tandem repeats, both with 8, and lastly, reverse repeat sequences with 2. We screened 56 SSR loci from the Grevillea robusta chloroplast genome, among which the single nucleotide repeat types were the most (37), and were mainly A/T sequence repeats, which accounted for about 62.50% of the total number of SSRs; there were three dinucleotide repeat types, two trinucleotide repeats, eight tetranucleotide repeats, five pentanucleotide repeats, and one hexanucleotide sequence type.

1.3 Boundary Characterization of the Chloroplast Genomes of Proteaceae

There are four boundaries in the tetrad structure of the chloroplast genome, namely, JLB(LSC/IRb), JSB(IRb/SSC), JSA(SSC/IRa), and JLA(IRa/LSC).Expansion and contraction phenomena of the IR region are very common in the process of the evolution of the chloroplast genome. In the chloroplast genomes of the six species of Proteaceae, the lengths of the IR regions were similar, the types of boundary genes were not significantly different, and the boundary genes of Grevillea robusta were similarly located to those of the four species of Macadamia and Helicia, which suggests that the IR regions of the chloroplast genome of Grevillea robusta did not undergo drastic contraction/expansion events, however, Protea kilimandscharica IRa/LSC boundary gene, trnH, entered the IRa region and was found to be in the IRa/LSC region. the IRa region and generated another copy of trnH in the IRb region. The IR region of Protea kilimandscharica underwent a small expansion event compared to the other five species of Proteaceae.

1.3 Analysis of gene selection pressures

The chloroplast protein coding genes of Grevillea robusta with Helicia shweliensis, Macadamia integrifolia and Protea kilimandscharica were analyzed for gene selection pressure by PAML software, and the results showed that rpl22 and matK had high ka values, and rpl33, ndhF, rpl22 and psaC had relatively high ks values. The Ka/Ks values of many genes are less than 1. It is evident that most of the protein-coding genes are in a state of purifying selection during the course of evolution. However, two genes (ycf1 and ycf2) had Ka/Ks values greater than 1, with Ka/Ks = 1.30 for ycf1 and 1.03 for ycf2. This suggests that they may have been positively selected during evolution.

1.4 RNA editing sites

The statistics of RNA editing sites are shown in (Fig. 5a). In the protein coding sequences of the Grevillea robusta chloroplast genome, a total of 148 RNA editing sites were detected for 42 protein coding genes, among which ndhB possessed the most editing sites (18), followed by psbA (13). In terms of editing efficiency, the number of sites possessing 100% editing efficiency reached 25, and the number of other low-frequency editing sites was also high (Fig. 5b), with a total of 47 sites with editing efficiency of less than 10%. In terms of editing types, all 12 possible editing types were found (Fig. 5c), among which, the highest number of C/U editing was found, accounting for 54.73% of the total RNA editing sites, i.e., 81 sites were edited from base C of the genome to base U of the mRNA strand, followed by T/C, i.e., 17 sites were edited from base T of the genome to base C of the mRNA strand.

1.5 Phylogenetic analysis

We selected representatives at the level of Proteales and constructed a phylogenetic tree based on the entire plastid genome sequence. The results showed that the four families in the order Proteales ( Proteaceae, Sabiaceae, Nelumbonaceae, and Platanaceae) were well clustered. Among them, Sabiaceae is at the most basal position and is the earliest one to be differentiated, followed by Nelumbonaceae. Proteaceae and Platanaceae were closer in affinity and had high support for each node. In the Proteaceae, Grevillea is in the same branch as Macadamia and Helicia with 100% support. Due to the scarcity of chloroplast genome resources in the Proteaceae, we were unable to make more in-depth and specific comparisons of the phylogenetic relationships of Grevillea robusta with species of other genera in the family.

Chloroplast genome sequences evolve at a fast rate, but their gene content and gene order are relatively highly conserved in green plants, making chloroplast genomes ideal for studying the phylogenetic and evolutionary relationships in plants.IR regions are usually present in the chloroplast genomes of most angiosperms, but there are exceptions, such as Fabaceae, where about 95% of the chloroplast genome lacks IR regions ( or only very short residues are present), and some species of the Cactaceae family have also been shown to lack IR regions, which have been interpreted as a novel evolutionary and structural change (Lavin et al. 1990; Sanderson et al. 2015). In this study, none of the IR regions of the Proteaceae species analyzed showed deletions and there were no significant differences in their lengths, which were relatively conserved in all species except for Protea kilimandscharica, which had a relatively small expansion of their IR regions. Changes at the junction of IR regions of chloroplast genomes can help to understand the evolutionary patterns of species (Tillich et al. 2017), and studies have shown that species with a high degree of similarity in the boundary region have a lower degree of differentiation at the chloroplast boundaries (Liu et al. 2018), therefore, from the results of the IR boundary analysis, it can be found that the Grevillea robusta is more closely related to Macadamia and Helicia .

SSRs in plant chloroplasts have been widely used in the study of plant population genetics and evolution and are thought to occur frequently in the non-coding regions of the chloroplast genome, especially in the gene spacer region (Singh et al. 2015). In this paper, a total of 56 SSRs of Grevillea robusta were screened, among which the single nucleotide repeat type was the most abundant, accounting for 66.07% of the total SSRs, and was mainly dominated by A/T. In this paper, four types of oligonucleotide repeats were screened: palindromic repeats, inverted repeats, tandem repeats and scattered repeats. Among them, the proportion of palindromic repeat sequences is as high as 47%. Oligonucleotide repeat sequences are used as a resource for polymorphic region identification when there are fewer resources for mutation hotspots in the chloroplast genome (Ahmed et al. 2012). Oligonucleotide repeats in Grevillea robusta play an important role in the evolution of the genome and will provide a theoretical basis for its molecular marker development, phylogeny of the Proteaceae or species identification.

The calculation of non-synonymous substitutions (Ka) and synonymous substitutions (Ks) can be used to detect gene conservation, and they are important for understanding the evolutionary dynamics of protein coding sequences and reconstructing the phylogeny of closely related species. According to the results of gene selection pressure analysis, most of the chloroplast protein-coding genes of Grevillea robusta and Helicia shweliensis, Macadamia integrifolia and Protea kilimandscharica had low Ka/Ks ratios (< 0.5), suggesting that these genes are under purifying selection. However, ycf1 had a high Ka/Ks ratio of 1.30, suggesting that the ycf1 gene may be under low selection pressure or higher positive selection conditions. ycf1 genes are DNA barcoding candidates in many species (Jia et al. 2017). The ycf2 gene of Grevillea robusta has Ka/Ks = 1.03, a value that is more likely to be considered as a sign that the gene was subjected to neutral selection rather than positive selection. Strong purifying selection pressure on genes can lead to higher chances of synonymous substitutions arising than non-synonymous ones (Lawrie et al. 2013). In the chloroplast genome of Grevillea robusta, we found that synonymous substitutions were more common than nonsynonymous substitutions, and other studies have found this trend in chloroplast genomes (Tae et al. 2014; Xu et al. 2015), which further supports our results.

In this study, a phylogenetic tree was constructed based on the chloroplast genomic data, and the support rate of each of its branches was high, indicating the reliability of the phylogenetic results, which provided strong evidence for the phylogenetic classification of Proteaceae and the understanding of the position of Grevillea robusta in Proteaceae. Although the development of high-throughput technology has brought the phylogenetic study to the genomic level, the current chloroplast genomic data are still relatively scarce for the species of Proteaceae, and as the study of the medicinal value of the species of Proteaceae gradually deepens, more genomic information is still needed to explore the phylogenetic relationships of the family in the future.

In this paper, the whole chloroplast genome of Grevillea robusta was sequenced, assembled and annotated, and its structure, gene composition, repeat sequences and RNA editing sites of protein-coding genes were analyzed. The total length of the chloroplast genome of silver birch was 158,642 bp, 129 genes were annotated, 56 SSR sites were screened, and 148 RNA editing events located in the protein coding sequences of chloroplasts were identified. Meanwhile, a comparative analysis between Grevillea robusta and four genera of Proteaceae revealed that they were not significantly different in terms of chloroplast genome length, gene number and composition, and the IR region of Grevillea robusta did not undergo any obvious contraction/expansion events as revealed by the boundary analysis. The results of phylogenetic analysis indicated that Grevillea was more closely related to the genera Macadamia and Helicia .Grevillea robusta has a high potential for exploitation both as timber and edible nuts. The sequencing and analysis of its chloroplast genome not only provides genetic resources for this important forest tree, but also provides a data base for the phylogenetic analysis of Proteaceae.

Data availability

All data generated or analyzed during this study are included in this published article.

Acknowledgements

Thanks to Mr. Zuo Youwei from Southwest University for their help.

Funding

The authors did not receive support from any organization for the submitted work.

Author information

Authors and Affiliations

College of Biological and Food Engineering, Huaihua University, Huaihua, Hunan, 418000, China.

Jiaxi Li

Key Laboratory of Research and Utilization of Ethnomedicinal Plant Resources of Hunan Province, Huaihua University, Huaihua, Hunan,418008, China.

Guanghua Liu

School of life sciences, Southwest University, Chongqing, 400715, China.

Yubing Yang

College of Horticulture and Landscape Architecture, Southwest University, Chongqing, 400715, China.

Jie Yu

Contributions

All the authors participated in compiling and analyzing the data and preparing the manuscript.

Corresponding author

Correspondence to guanghualiu

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Conflict of interest

The authors declare no conflict of interest.

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Characteristics of Complete Chloroplast Genome of Grevillea robusta

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