Size and structure of chloroplast genome.
The complete chloroplast genome of D. yangii is 95,156 bp with two inverted repeats of 22,515 bp separated by a large single-copy region (43,828 bp) and a small single-copy region (6,298 bp). The GC content is highest in IR (~ 43.9%) and lowest in the SSC (~ 28.8%, Table 1).
Table 1
Base composition in each part in chloroplast genome of D. yangii
| A(%) | C(%) | G(%) | T(%) | GC(%) | Length(bp) |
LSC | 34.3 | 15.8 | 15.6 | 34.3 | 31.4 | 43828 |
IR | 28 | 22.4 | 21.5 | 28 | 43.9 | 22515 |
SSC | 30.4 | 14.9 | 13.9 | 40.8 | 28.8 | 6298 |
Total | 31.1 | 18.6 | 18.5 | 31.8 | 37.1 | 95156 |
The CP genome encodes 90 genes, of which 11 genes (including rpl2, rpl23, rps7, rps19, ycf1, ycf2, ycf15, rrn16, rrn23, rrn4.5, rrn5) are repeated in the IR region. The other 79 genes are composed of 42 protein coding genes, 31 tRNAs, 4 rRNAs, and 2 pseudogenes (petB, ycf1). Among them, the six genes with introns are clpP, rps12, rpl2, rpl16, rps16, and trnV-UAC (Table 2 and Fig. 1).
Genes inside outer ring are transcribed clockwise, while genes outside outer ring are transcribed counterclockwise. Genes of different functional groups are shown in colored bars. The inner circle (dashed gray area) indicates the proportional GC content of the corresponding genes. Regions of the large single-copy (LSC), small single-copy (SSC) and inverted repeats (IRA and IRB) are indicated.
Table 2
List of annotated genes in D. yangii
Category of genes | Gene names |
Ribosomal RNA genes | rrn16、rrn23、rrn4.5、rrn5 |
Transfer RNA genes | trnC-GCA、trnD-GUC、trnE-UUC、trnF-GAA |
| trnfM-CAU、trnH-GUG、trnH-GUG、trnI-CAU |
| trnI-CAU、trnI-GAU、trnI-GAU、trnK-UUU |
| trnL-CAA、trnL-CAA、trnM-CAU、trnN-GUU |
| trnN-GUU、trnP-UGG、trnQ-UUG、trnR-ACG |
| trnR-ACG、trnR-UCU、trnS-GGA、trnS-UGA |
| trnT-GGU、trnT-UGU、trnV-GAC、trnV-GAC |
| trnV-UAC、trnW-CCA、trnY-GUA |
Small subunit of ribosome | rps2、rps3、rps4、rps7、rps8、rps11、rps12 |
| rps14、rps15、rps16、rps18、rps19 |
Large subunit of ribosome | rpl2、rpl14、rpl16、rpl20、rpl22、rpl23、rpl32、rpl33、rpl36 |
DNA dependent RNA polymerase | / |
Translational initiation factor | infA |
Subunits of photosystem I | psaJ |
Subunits of photosystem Ill | psbH、psbJ、psbK、pabM、psbN、pabT、pabZ |
Subunits of Cytochrome | petA、petB、petG、petL、petN |
Subunits of ATP synthase | atpE、atpI |
ATP-dependent protease | clpP |
eLarge subunit of Rubisco | / |
Subunits of NADH | / |
maturase | matK |
Envelop membrane protein | cema |
Subunit of Acetyl-CoA-carboxylase | accD |
c-type cytochrome synthesis gene | / |
Conserved Open Reading Frames | ycf1、ycf2、ycf15 |
Basic characteristic of CP genome in Calypsoinae.
Calypsoinae contains 79 species of 12 genera, including 17 mycoheterotrophic species related to three heterotrophic genera (Corallorhiza, Danxiaorchis and Yoania). Seven genera were selected in this study, representing autotrophic (11) and heterotrophic (14) orchids to explore sequence and structural variation of chloroplast genome. There are typical tetrad structures in the chloroplast genome of both autotrophic and mycoheterotropic orchids from Calypsoinae, which differs from those of heterotrophic orchids, wherein the chloroplast genome of Gastrodia elata does not have a typical tetrad structure due to loss of IR region [16,17]. There were no significant differences in CP genome size between autotrophic and heterotrophic orchids, ranging from 142 kb to 160 kb and 88 kb to 151 kb, respectively, with larger fluctuations in heterotrophic. The smallest chloroplast genome was D. singchiana (88 kb) and, notably, Cremastra unguiculata (160 kb) was twice as large as D. singchiana. The CP genome size was below 100 kb in Danxiaorchis, while it ranged from 120 to160 kb in Corallorhiza, which may be related to the apparently smaller LSC region of the Danxiaorchis genus (Table 3).
Furthermore, the GC contents of autotrophic orchids ranged from 34.54%. to 37.2%, which is not significantly different from 25.8–37.2% deteceted in heterotrophic orchids. However, the GC content of Corallorhiza striata var. involute was 25.8%, versus 37.4% in T. josephii and T. szechuanica. In this study, variations were detected at CP genomes with different nutritional types, and they presented gene loss phenomena, specifically of ndh genes, except from Changnienia amoena, Corallorhiza bulbosa, Cremastra unguiculata and O. foliosa. Most photosynthesis-related genes were missing or pseudogenenized, such as various degree of absence in the photosystem Ⅰ and II (psa/psb), ndh, and atp genes; only housekeeping genes were preserved (Fig. 2).
Table 3 Chloroplast genomic characteristics of 25 species of Calypsoinae
Species
|
GenBank no.
|
Length (bp)
|
LSC (bp)
|
SSC (bp)
|
IR (bp)
|
GC %
|
coding genes
|
tRNAs
|
rRNAs
|
pseudogenes
|
Changnienia amoena#
|
MN047293
|
156,818
|
84,847
|
18,141
|
26,915
|
37.1
|
87
|
37
|
8
|
0
|
Calypso bulbosa#
|
MN990433
|
149,451
|
83,800
|
14,343
|
25,654
|
37.1
|
76
|
38
|
8
|
5
|
Calypso bulbosa var. Occidentalis#
|
NC040980
|
149,313
|
83,331
|
14,718
|
25,632
|
37.1
|
74
|
38
|
8
|
9
|
Corallorhiza bentleyi*
|
MG874035
|
124,482
|
63,592
|
7,770
|
26,560
|
36.6
|
60
|
39
|
8
|
3
|
Corallorhiza bulbosa*
|
NC025659
|
148,643
|
82,851
|
12,368
|
26,712
|
37.1
|
74
|
38
|
8
|
9
|
Corallorhiza trifida*
|
MN990435
|
149,408
|
83,171
|
14,397
|
25,920
|
37.2
|
74
|
38
|
8
|
9
|
Corallorhiza macrantha*
|
NC025660
|
151,031
|
84,263
|
12,544
|
27,112
|
37.2
|
72
|
38
|
8
|
11
|
Corallorhiza maculata var.mexicana*
|
KM390015
|
151,506
|
84,347
|
12,671
|
27,244
|
37.1
|
73
|
38
|
8
|
1
|
Corallorhiza maculata var.occidentalis*
|
KM390016
|
146,595
|
81,363
|
12,368
|
26,432
|
36.9
|
60
|
38
|
8
|
16
|
Corallorhiza mertensiana*
|
NC025661
|
147,941
|
81,109
|
13,774
|
26,529
|
36.8
|
60
|
38
|
8
|
25
|
Corallorhiza odontorhiza*
|
NC025664
|
147,317
|
82,257
|
13,508
|
25,776
|
37
|
73
|
38
|
8
|
10
|
Corallorhiza striata var. involuta*
|
MG874038
|
124,433
|
63,680
|
7,653
|
26,550
|
25.8
|
64
|
38
|
8
|
0
|
Corallorhiza striata var. striata*
|
MG874034
|
141,202
|
75,779
|
13,101
|
26,161
|
27.3
|
55
|
37
|
8
|
20
|
Corallorhiza striata var.vreelandii*
|
JX087681
|
137,505
|
72,631
|
12,388
|
26,243
|
36.4
|
48
|
37
|
8
|
24
|
Corallorhiza wisteriana*
|
NC025663
|
146,437
|
76,350
|
17,743
|
26,172
|
37.1
|
73
|
38
|
8
|
10
|
Cremastra appendiculata#
|
NC037439
|
155,320
|
87,098
|
15,478
|
26,372
|
37.2
|
79
|
38
|
8
|
6
|
Cremastra unguiculata#
|
MN200381
|
159,341
|
87,031
|
18,252
|
27,029
|
36.9
|
85
|
38
|
8
|
1
|
Danxiaorchis yangii*
|
newly sequenced
|
95,156
|
43,828
|
6,298
|
22,515
|
37.1
|
49
|
31
|
8
|
2
|
Danxiaorchis singchiana*
|
MN584923
|
87,931
|
42,575
|
17,831
|
13,762
|
34.54
|
35
|
28
|
4
|
21
|
Oreorchis angusta#
|
MN990443
|
158,654
|
86,294
|
18,312
|
27,024
|
37
|
86
|
38
|
8
|
0
|
Oreorchis foliosa#
|
MN990441
|
158,496
|
86,061
|
18,361
|
27,037
|
36.9
|
82
|
38
|
8
|
4
|
Oreorchis foliosa var. indica#
|
MN990440
|
158,599
|
86,109
|
17,616
|
27,437
|
36.9
|
82
|
38
|
8
|
4
|
Oreorchis patens#
|
MN990436
|
158,256
|
86,253
|
18,341
|
26,831
|
35
|
86
|
38
|
8
|
0
|
Tipularia josephii#
|
MN990437
|
146,816
|
82,881
|
11,981
|
25,977
|
37.4
|
74
|
38
|
8
|
7
|
Tipularia szechuanica#
|
MN990442
|
142,799
|
82,378
|
11,533
|
24,444
|
37.4
|
74
|
38
|
8
|
7
|
Agrostophyllum callosum#
|
MN990432
|
158,365
|
86,503
|
18,376
|
26,743
|
29.9
|
86
|
38
|
8
|
0
|
Note: * stand for mycoheterotrophy; # stand for autotrophy.
Collinearity Analysis Of Chloroplast Genome In Calypsoinae
This investigation conducted collinearity analysis of CP genome of 25 species with Agrostophyllum callosum as the reference, and the result show the plastid genome of autotrophic orchids is completely collinear, without rearrangements or inversions. However, such changes did exist in D. yangii and D. singchiana, including inversions of IR and SSC within D. singchiana, as well as rearrangements and inversions of LSC in D. yangii. Moreover, there are no rearrangements or inversions detected in Corallorhiza (Fig. 3).