Basic structural features of the chloroplast genome
The chloroplast genome sequences of the four species of Dendrobium were 152252bp (D. flexicaule), 152226bp (D. officinale), 151281bp (D. huoshanense), and 152130bp (D. moniliforme). All of them have a circular retrad structure, including alarge single copy, a small single copy and two inverted repeat regions. Among the four sections, the highest GC content is in the IR region (43.4%), and the lowest GC content is in the SSC region (30.3%-30.5%) (Fig. 1). The full-length chloroplast genome sequence of D. flexicaule is 152252bp, and the GC content is 37.5%. The total of 122 genes were annotated in the chloroplast genome of D. flexicaule, including 77 PCGs, 37 tRNA genes, and 8 rRNA genes. The tRNA genes and rRNA genes of the four chloroplast genomes were the same, but there were certain differences in the number of PCGs. Compared with D. huoshanense and D. moniliforme, D. flexicaule and D. officinale lack four protein-coding genes, (Table. 1).
Table 1
Comparison of chloroplast genome structure characteristics of four Dendrobium species
Genomic features
|
flexicaule
|
D.
officinale
|
D. huoshanense
|
D. moniliforme
|
Total genome length/bp
|
152252
|
152226
|
151281
|
152130
|
LSC region length/bp
|
85049
|
85096
|
84888
|
85192
|
Length of SSC region/bp
|
14495
|
14514
|
14005
|
14352
|
IR region length/bp
|
26354
|
26308
|
26194
|
26293
|
Total number of genes
|
122
|
122
|
118
|
118
|
Number of PCGs
|
77
|
77
|
73
|
73
|
Number of tRNA genes
|
37
|
37
|
37
|
37
|
Number of rRNA genes
|
8
|
8
|
8
|
8
|
GC content in the LSC region
|
37.5
|
37.5
|
37.5
|
37.5
|
GC content in the LSC fegion
|
35.0
|
35.0
|
35.1
|
35.1
|
GC content in the SSC region
|
30.3
|
30.3
|
30.3
|
30.5
|
GC content in the IR region
|
43.4
|
43.4
|
43.4
|
43.4
|
Codon Usage Analysis
There were 64 codons (including 3 stop codons) encoded by the 4 species of Dendrobium cp genome, encoding a total of 50427–50750 amino acids (including terminators), among which the number of codons encoded by D. flexicaule is the largest, and the number of D. huoshanense is the least. 61 codons encoded a total of 20 amino acids, among which the number of codons encoding leucine is the largest and tryptophan is the least. D. flexicaule encoded a total of 50,750 amino acids, including 4,978 leucine and 673 tryptophan. There are 30 codons with RSCU values > 1, 26 codons ending in A/U, 31 codons < 1, and 26 codons ending in C/G, which indicated that D. flexicaule cp genome had a higher preference for codons ending in A/U. From the codon RSCU value map (Fig. 2), the codon AGA of the four species of Dendrobium encoding arginine had the strongest preference (the highest RSCU value of D. flexicaule: 2.21, and the lowest RSCU value of D. moniliforme: 2.11).
Ir Boundary Expansion And Contraction
The chloroplast genome is a circular structure composed of four regions: SSC, IRb, SSC and IRa. Among them, the IR region is relatively conserved. The phenomenon of contraction and expansion of the IR region represents the evolution of plants, and the size of the chloroplast genome of different plants is also related to it (Hui et al. 2014). In this study, the IR boundaries of D. flexicaule and its three related species were analyzed and compared (Fig. 3). The LSC/IRb and IRa/LSC boundaries were relatively conservative. The rpl22 gene was located in the LSC/IRb boundary, and the sequence length in the IRb region was all 37 bp. The four Dendrobium IRa/SSC boundaries were all located between rsp19 and psbA. The IR/SSC boundary was quite different. The ycf1 gene stretched across the IRa/SSC boundary, resulting in an incomplete duplication ofΨycf1 in the IRb region. In addition, the IRb/SSC node of D. flexicaule and D. moniliforme expanded into the ndhF gene, resulting in 10bp and 199bp overlap between ndhF and ycf1 and expanding into the IR region. But the ndhF genes of D. officinale and D. huoshanense were located in the SSC region, and the distances from the IRb/SSC boundary were 4bp and 1bp.
Simple Sequence Repeat Identification
Simple repeats, usually with 1–6 base tandem repeats as microsatellite sequences, are rich in polymorphisms and are highly efficient molecular markers that were often used in species identification (Wu et al. 2020). Through the analysis of four chloroplast genomes (Table 2), all four types of Dendrobium cover 6 types of nucleotide sequences. 58, 58, 55, and 58 SSR sequences were detected from the chloroplast genomes of D. flexicaule, D. officinale, D. huoshanense, D. moniliforme, respectively, among which the number of mono-nucleotide repeats was the largest (accounting for 58.62%-60.34%), And all were A/T repeats, followed by di-nucleotide repeats (20.0%-22.4%), tetra-nucleotides (6.90%-12.73%), tri-nucleotide repeats (1.82%-6.90%), penta-nucleotide and hexa-nucleotide repeat types were rare. The distribution of SSR sequences in various regions was analyzed, and it was found that SSR was mainly distributed in the LSC region, and the distribution ratios of the four Dendrobium species were 70.69%, 68.97%, 63.64% and 65.52%, respectively. The highest proportion of the IR region is D. huoshanense(27.27%), and the lowest is D. flexicaule and D. officinale (13.79%). The highest proportion of SSC region is D. officinale (17.24%), and the lowest is D. huoshanense(9.09%),( Fig. 4).
Table 2
Statistics of SSR information of four kinds of Dendrobium chloroplast genomes
SSR type
|
Nucleotide type
|
D.
flexicaule
|
D.
officinale
|
D. huoshanense
|
D. moniliforme
|
single nucleotide
|
A/T
|
34
|
34
|
33
|
33
|
C/G
|
-
|
-
|
-
|
2
|
total
|
34
|
34
|
33
|
35
|
dinucleotide
|
AG/CT
|
2
|
2
|
2
|
2
|
AT/AT
|
11
|
12
|
9
|
11
|
total
|
13
|
14
|
11
|
13
|
trinucleotide
|
AAT/ATT
|
3
|
3
|
-
|
2
|
ACT/AGT
|
1
|
1
|
-
|
-
|
AGG/CCT
|
-
|
-
|
1
|
-
|
total
|
4
|
4
|
1
|
2
|
tetranucleotides
|
AAAG/CTTT
|
2
|
1
|
4
|
3
|
AATT/AATT
|
1
|
1
|
1
|
1
|
ACAG/CTGT
|
1
|
1
|
1
|
1
|
AGAT/ATCT
|
1
|
1
|
1
|
1
|
total
|
5
|
4
|
7
|
6
|
pentanucleotide
|
AATAG/ATTCT
|
-
|
1
|
-
|
-
|
AAAAG/CTTTT
|
-
|
-
|
1
|
-
|
ATATC/ATATG
|
-
|
-
|
-
|
1
|
AATCC/ATTGG
|
-
|
-
|
1
|
-
|
total
|
0
|
1
|
2
|
1
|
hexanucleotide
|
AAAAAG/CTTTTT
|
1
|
-
|
1
|
-
|
ACAGAT/ATCTGT
|
1
|
-
|
-
|
-
|
AGATGG/ATCTCC
|
-
|
1
|
-
|
1
|
total
|
2
|
1
|
1
|
1
|
total
|
|
58
|
58
|
55
|
58
|
Chloroplast Genome Sequence Comparison And Phylogeny
In order to compare the chloroplast genome sequence of D. flexicule with the other three species of Dendrobium. Taking D. flexicaule as a referencethe online tool mVISTA was used to compare the entire sequence with D.officinale, D.huoshanense, and D. moniliforme . (Fig. 5). The four chloroplast genome sequences showed that the variation of protein coding region was lower than that of non-coding region, the variation region was mainly concentrated in LSC region and SSC region, and the variation of IR region was lower than that of SSC and LSC region. It can be seen from the figure that the genes with large variation between D. fleixicaule and the other three species of Dendrobium were: ndhF and ycf1. Most genes were maintained at 90% similarity, and there were more inter-gene variations than gene regions, such as: matK-trnK-UUU, rps16-trnQ-UGG, petN-psbM, trnT-UGU-trnL-UAA, accD-psal, clpP-psbB, psbB-psbT, ndhF-rpl32, etc.
In addition, the software DnaSP6 tool was used to calculate the nucleotide variation value (Pi) between the chloroplast genomes, and the cp genome hypervariable regions were analyzed. (Fig. 6). Nucleotide variations in the chloroplast genomes of four species of Dendrobium were mainly concentrated in the SSC and LSC regions. The Pi value is generally below 0.25. Combined with the mVISTA map, the top 5 nucleotide variation values were screened out, which were ndhF-rpl32 (SSC region), ycf1 (SSC region-IRa region), accD-psal (LSC region), rpl16-trnQ-UUG (LSC region), trnT-GGU-psbD (LSC region).
In this study, the complete cp genome sequences of 11 species of Dendrobium species were downloaded from the NCBI database and compared with the complete cp genome sequences of D. flexicaule, among which Phalaenopsis aphrodite and P. aphrodite were the outgroups. Aligned sequences were used MEGA software to construct the Maximum Likehood phylogenetic tree(Fig. 7). Twelve species of Dendrobium were clustered together, and two species of Phalaenopsis were clustered together, D. officinale and D. flexicaule were first grouped into a branch, and the support rate was 100% at the level of 100 inspections, and then gather together with D. aduncum into a branch, with a support rate of 100%. D. huoshanense and D.monilifore grouped into one branch. It showed that D. officinale and D. flexicaule were closely related, and D.huoshanense and D.monilifore were closely related.