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Complete Chloroplast Genome and Comparative Analysis of Three Lycium (Solanaceae) Species with Medicinal and Edible PropertiesYingxian Cui 1,3, Jianguo Zhou 1,3, Xinlian Chen 1,3, Zhichao Xu 1,3, Yu Wang 1,3, Wei Sun 2, Jingyuan Song 1,3, and Hui Yao 1,3,*

1 Key Lab of Chinese Medicine Resources Conservation, State Administration of Traditional Chinese Medicine of the People's Republic of China, Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, Beijing, 100193, China; [email protected] (Y.C.); [email protected] (J.Z.); [email protected] (X.C.); [email protected] (Z.X.); [email protected] (Y.W.); [email protected] (J.S.)

2 Key Lab of Beijing for Identification and Safety Evaluation of Chinese Medicine, Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing, 100700, China; [email protected] (W.S.)

3 Engineering Research Center of Chinese Medicine Resources, Ministry of Education, Beijing 100193, China;

* Correspondence: [email protected] ; Tel.: +86-10-57833194

Figure S1. Phylogenetic tree constructed using maximum likelihood (ML) method

based on the complete chloroplast genomes from different species. Numbers at

nodes are values for bootstrap support.

A. Lycium barbarum_LSC-IRa_F:

B. Lycium barbarum_IRa-SSC_F:

C. Lycium barbarum__SSC-IRb_R:

D. Lycium barbarum__IRb-LSC_R:

Figure S2. Peak map of Sanger sequencing.

Table S1. Primer sequences at the accD gene of L. ruthenicum.

Species Regions Forward/Reverse Primer sequence (5' to 3')

L.ruthenicuminsertion

F TCCCCTTTGTTCCGTGTTGAR TCCGAATGTCTTTAACGCCG

deletionF TACGACATAGGAGAAGCCGC

R AGTAGGGATCTTCGCCCCTA

Table S2. Genes with introns in the chloroplast genomes of L. barbarum, L. chinense , and L. ruthenicum as well as the lengths of the exons and introns.

Species Gene Location Exon I (bp)

Intron I (bp)

Exon II (bp)

Intron II (bp)

Exon III (bp)

L. barbarum atpF LSC 144 799 411

clpP LSC 69 844 296 630 253

ndhA SSC 556 1050 539 　　ndhB IR 777 700 756

petB LSC 6 872 642

petD LSC 8 749 475

rpl16 LSC 9 1049 402

rpl2 IR 391 662 431

rpoC1 LSC 432 723 1638

rps7 IR 231 551 27 　　rps12* LSC 114 - 231 553 27

rps16 LSC 40 727 218 　　trnA-UGC IR 38 801 35

trnG-UCC LSC 23 702 47

trnI-GAU IR 42 935 35

trnK-UUU LSC 37 2688 35

trnL-UAA LSC 35 532 50

trnV-UAC LSC 38 604 37

trnV-GAC IR 27 551 231 　　ycf3 LSC 127 715 230 777 153

L. chinense atpF LSC 145 702 410

clpP LSC 71 794 291 639 229ndhA SSC 553 1154 539ndhB IR 777 679 756petB LSC 6 750 642petD LSC 8 742 475rpl16 LSC 9 1016 396rpl2 IR 393 663 435rpoC1 LSC 453 737 1614rps12* LSC 114 - 232 536 26rps16 LSC 41 822 226trnA-UGC IR 38 681 35trnG-UCC LSC 23 692 48trnI-GAU IR 42 711 35trnK-UUU LSC 37 2513 35trnL-UAA LSC 37 495 50trnV-UAC LSC 38 563 37

ycf3 LSC 125 743 229 757 153

L. ruthenicum atpF LSC 145 695 410

clpP LSC 71 793 291 636 229ndhA SSC 553 1154 539 　ndhB IR 777 679 756

petB LSC 6 743 642

petD LSC 8 742 475

rpl16 LSC 9 1018 396

rpl2 IR 393 663 435

rpoC1 LSC 453 741 1614

rps12* LSC 114 - 232 536 26

rps16 LSC 41 822 226 　trnA-UGC IR 38 681 35

trnG-UCC LSC 23 693 48

trnI-GAU IR 42 711 35

trnK-UUU LSC 37 2509 35

trnL-UAA LSC 37 495 50

trnV-UAC LSC 38 563 37

ycf3 LSC 125 741 229 760 153

* The rps12 gene is a trans-spliced gene with the 5’ end located in the LSC region and the duplicated 3’ ends in the IR regions.

Table S3. Codon-anticodon recognition pattern and codon usage for the chloroplast genomes of L. barbarum, L. chinense, and L. ruthenicum.

species Amino acid Codon No. RSCU tRNA Amino acid Codon No. RSCU tRNA

L.barbarum Phe UUU 957 1.29 Tyr UAU 769 1.61

Phe UUC 531 0.71 trnF-GAA Tyr UAC 188 0.39 trnY-GUA

Leu UUA 856 1.83 trnL-UAA Stop UAA 46 1.6

Leu UUG 576 1.23 trnL-CAA Stop UAG 22 0.77

Leu CUU 612 1.31 His CAU 476 1.55

Leu CUC 201 0.43 His CAC 138 0.45 trnH-GUG

Leu CUA 375 0.8 trnL-UAG Gln CAA 712 1.5 trnQ-UUG

Leu CUG 192 0.41 Gln CAG 239 0.5

Ile AUU 1092 1.47 Asn AAU 979 1.52

Ile AUC 455 0.61 trnI-GAU Asn AAC 312 0.48 trnN-GUU

Ile AUA 680 0.92 trnI-CAU Lys AAA 1037 1.47 trnK-UUU

Met AUG 619 1 trn(f)M-CAU Lys AAG 371 0.53

Val GUU 524 1.46 Asp GAU 865 1.6

Val GUC 183 0.51 trnV-GAC Asp GAC 214 0.4 trnD-GUC

Val GUA 536 1.49 trnV-UAC Glu GAA 1023 1.47 trnE-UUC

Val GUG 194 0.54 Glu GAG 366 0.53

Ser UCU 591 1.71 Cys UGU 218 1.45

Ser UCC 334 0.96 trnS-GGA Cys UGC 82 0.55 trnC-GCA

Ser UCA 419 1.21 trnS-UGA Stop UGA 18 0.63

Ser UCG 197 0.57 Trp UGG 475 1 trnW-CCA

Pro CCU 438 1.57 Arg CGU 349 1.3 trnR-ACG

Pro CCC 204 0.73 Arg CGC 101 0.38

Pro CCA 321 1.15 trnP-UGG Arg CGA 393 1.47

Pro CCG 154 0.55 Arg CGG 116 0.43

Thr ACU 524 1.56 Arg AGA 411 1.19 trnR-UCU

Thr ACC 269 0.8 trnT-GGU Arg AGG 126 0.36

Thr ACA 407 1.21 trnT-UGU Ser AGU 477 1.78

Thr ACG 145 0.43 Ser AGC 173 0.65 trnS-GCU

Ala GCU 620 1.77 Gly GGU 565 1.25

Ala GCC 247 0.7 Gly GGC 207 0.46 trnG-GCC

Ala GCA 393 1.12 trnA-UGC Gly GGA 722 1.59 trnG-UCC

Ala GCG 145 0.41 Gly GGG 320 0.71

L.chinense Phe UUU 958 1.29 Tyr UAU 766 1.61




Leu CUU 610 1.3 His CAU 477 1.55



Leu CUG 192 0.41 Gln CAG 238 0.5

Ile AUU 1094 1.47 Asn AAU 979 1.52




Val GUU 524 1.46 Asp GAU 865 1.6



Val GUG 194 0.54 Glu GAG 365 0.53

Ser UCU 594 1.71 Cys UGU 218 1.45





Pro CCC 205 0.73 Arg CGC 101 0.38


Pro CCG 155 0.56 Arg CGG 117 0.44





Ala GCU 622 1.77 Gly GGU 564 1.24



Ala GCG 140 0.4 Gly GGG 320 0.71

L.ruthenicum Phe UUU 940 1.28 Tyr UAU 751 1.61




Leu CUU 605 1.31 His CAU 465 1.55



Leu CUG 187 0.41 Gln CAG 235 0.5

Ile AUU 1065 1.46 Asn AAU 953 1.51




Val GUU 516 1.46 Asp GAU 840 1.6



Val GUG 191 0.54 Glu GAG 351 0.52

Ser UCU 577 1.71 Cys UGU 210 1.45





Pro CCC 198 0.72 Arg CGC 98 0.37


Pro CCG 154 0.56 Arg CGG 114 0.43





Ala GCU 613 1.76 Gly GGU 553 1.24



Ala GCG 140 0.4 Gly GGG 314 0.71

RSCU: Relative Synonymous Codon Usage.

Table S4. RNA editing predicted in the chloroplast genomes of three Lycium species by the PREP

program.

GeneNucleotide Position Codon

changeAmino acid

change Score① ② ③accD - - - - - -

atpA 791 791 791 CCC-CTC P-L 1

atpB - - - - - -

atpF 92 92 92 CCA-CTA P-L 0.86

atpI - - - - - -

ccsA - - - - - -

clpP - - - - - -

matK - - - - - -

ndhA 341 341 341 TCA-TTA S-L 1

566 566 566 TCA-TTA S-L 1

1073 1073 1073 TCC-TTC S-F 1

ndhB 149 149 149 TCA-TTA S-L 1

467 467 467 CCA-CTA P-L 1

586 586 586 CAT-TAT H-Y 1

611 611 611 TCA-TTA S-L 0.8

737 737 737 CCA-CTA P-L 1

746 746 746 TCT-TTT S-F 1

830 830 830 TCA-TTA S-L 1

836 836 836 TCA-TTA S-L 1

1481 1481 1481 CCA-CTA P-L 1

ndhD 2 2 2 ACG-ATG T-M 1

383 383 383 TCA-TTA S-L 1

674 674 674 TCG-TTG S-L 1

878 878 878 TCA-TTA S-L 1

1298 1298 1298 TCA-TTA S-L 0.8

- 1310 1310 TCA-TTA S-L 0.86

ndhF 290 290 290 TCA-TTA S-L 1

ndhG - - - - - -

petB 418 418 418 CGG-TGG R-W 1

611 611 611 CCA-CTA P-L 1

petD - - - - -

petG - - - - -

petL - - - - -

psaB - - - - -

psaI - - - - -

psbB - - - - - -

psbE 214 214 214 CCT-TCT P-S 1

psbF - - - - - -

psbL 2 2 2 ACG-ATG T-M 1

rpl20 308 308 308 TCA-TTA S-L 0.86

rpl23 - - - - - -

rpl2 - - - - - -

rpoA 830 830 830 TCA-TTA S-L 1

rpoB 338 338 338 TCT-TTT S-F 1

473 473 473 TCA-TTA S-L 0.86

551 551 551 TCA-TTA S-L 1

2000 2000 2000 TCT-TTT S-F 1

2426 2426 2426 TCA-TTA S-L 0.86

rpoC1 62 62 62 TCA-TTA S-L 1

rpoC2 2299 2299 2299 CGG-TGG R-W 1

3743 3743 3743 TCA-TTA S-L 0.86

rps14 80 80 80 TCA-TTA S-L 1

rps16 - - - - - -

rps2 248 248 248 TCA-TTA S-L 1

rps8 - - - - - -

ycf3 - - - - - -

①L.barbarum; ②L.chinense; ③L.ruthenicum

Table S5. Simple sequence repeats (SSRs) in the chloroplast genome of L. barbarum, L. chinense, and L.

ruthenicum.

specie SSR nr. SSR type SSR size start end

L.barbarum 1 p3 (AAG)4 12 2968 29792 p1 (T)11 11 3759 37693 p4 (TTCT)3 12 5464 54754 p2 (AT)6 12 6345 63565 p2 (AT)5 10 7024 70336 p4 (TTTG)3 12 7149 71607 p1 (A)11 11 8240 82508 p2 (AT)5 10 8746 87559 p1 (T)11 11 9551 956110 c (A)12(AT)5 116 9884 999911 p1 (T)12 12 12367 1237812 p4 (TTAT)3 12 14387 1439813 p1 (A)11 11 16572 1658214 p1 (T)10 10 18797 1880615 p1 (A)11 11 29827 2983716 p4 (CTAT)3 12 30173 3018417 p4 (TTTA)3 12 30657 3066818 p1 (T)10 10 33176 3318519 c (TATT)3(A)10 93 33581 3367320 p1 (T)10 10 36568 3657721 p4 (TATT)3 12 37723 3773422 p2 (TA)5 10 43468 4347723 p1 (T)16 16 44692 4470724 p1 (A)15 15 45677 4569125 p1 (T)10 10 45928 4593726 p2 (TA)5 10 47904 4791327 p3 (TTA)4 12 53293 5330428 c (T)10(AAAT)3 75 56203 5627729 p1 (A)11 11 56848 5685830 p1 (T)11 11 59004 5901431 p4 (CAAA)3 12 66246 6625732 p1 (T)10 10 69288 6929733 p2 (TA)5 10 70286 7029534 p4 (AAAC)3 12 70406 7041735 p1 (T)14 14 71473 7148636 p1 (T)12 12 72010 7202137 p1 (T)16 16 72963 7297838 p1 (A)12 12 73130 7314139 p1 (T)10 10 73978 7398740 p1 (A)10 10 76519 76528

41 p1 (T)10 10 80558 8056742 p1 (T)16 16 86616 8663143 p2 (AT)5 10 87257 8726644 p1 (A)15 15 109745 10975945 p1 (A)10 10 112009 11201846 p1 (A)10 10 114652 11466147 p1 (T)11 11 115460 11547048 p4 (TTGA)3 12 119290 11930149 p1 (A)10 10 121249 12125850 p1 (A)10 10 122202 12221151 p2 (AT)5 10 122930 12293952 p1 (T)10 10 127618 12762753 p1 (T)15 15 132452 13246654 p2 (AT)5 10 154945 15495455 p1 (A)16 16 155580 155595

L.chinense 1 p3 (AAG)4 12 2981 29922 p1 (T)15 15 3772 37863 p1 (A)11 11 4602 46124 p4 (TTCT)3 12 5477 54885 p2 (AT)6 12 6358 63696 p1 (T)14 14 6692 67057 p2 (AT)5 10 7043 70528 p4 (TTTG)3 12 7168 71799 p1 (T)11 11 9566 957610 p1 (A)19 19 9899 991711 p1 (T)12 12 12378 1238912 p4 (TTAT)3 12 14366 1437713 p1 (A)11 11 16567 1657714 p1 (T)10 10 18792 1880115 p1 (A)11 11 29832 2984216 p4 (CTAT)3 12 30178 3018917 p4 (TTTA)3 12 30662 3067318 p1 (T)11 11 31165 3117519 p1 (T)10 10 32894 3290320 c (TATT)3(A)10 98 33599 3369621 p1 (T)12 12 36591 3660222 p4 (TATT)3 12 37757 3776823 p2 (TA)5 10 43486 4349524 p1 (T)14 14 44716 4472925 p1 (A)15 15 45699 4571326 p2 (TA)5 10 47924 4793327 p3 (TTA)5 15 52706 5272028 p3 (TTA)4 12 53345 53356

29 c (T)10(AAAT)3 75 56254 5632830 p1 (A)12 12 56893 5690431 p1 (T)10 10 59050 5905932 p1 (T)10 10 65512 6552133 p4 (CAAA)3 12 66305 6631634 p1 (T)10 10 68475 6848435 p2 (TA)5 10 70321 7033036 p4 (AAAC)3 12 70441 7045237 p1 (T)18 18 71529 7154638 p1 (T)10 10 72070 7207939 p1 (T)17 17 73021 7303740 p1 (A)10 10 73189 7319841 p1 (T)15 15 74035 7404942 p1 (A)12 12 76577 7658843 p1 (T)10 10 80625 8063444 p1 (T)24 24 86658 8668145 p2 (AT)5 10 87307 8731646 p1 (A)12 12 109813 10982447 p4 (TTGA)3 12 119364 11937548 p2 (AT)5 10 122984 12299349 p1 (T)10 10 127686 12769550 p1 (T)12 12 132520 13253151 p2 (AT)5 10 155028 15503752 p1 (A)24 24 155663 155686

L.ruthenicum 1 p3 (AAG)4 12 2999 30102 p1 (T)11 11 3790 38003 p1 (A)10 10 4616 46254 p4 (TTCT)3 12 5492 55035 p2 (AT)6 12 6373 63846 p1 (T)15 15 6707 67217 p2 (AT)5 10 7059 70688 p4 (TTTG)3 12 7184 71959 p1 (A)11 11 8267 827710 p1 (A)10 10 8765 877411 p1 (T)12 12 9577 958812 c (T)10(A)17(AT)5 152 9881 1003213 p1 (T)13 13 12382 1239414 p1 (A)13 13 13065 1307715 p1 (A)10 10 16508 1651716 p1 (T)10 10 18732 1874117 p5 (AATTG)3 15 27243 2725718 p1 (A)10 10 29791 2980019 p4 (TTTA)3 12 30616 30627

20 p1 (T)10 10 31119 3112821 p1 (A)10 10 32213 3222222 p1 (T)10 10 32540 3254923 p1 (T)10 10 32713 3272224 c (TATT)(A)10 93 33118 3321025 p1 (T)11 11 36112 3612226 p4 (TATT)3 12 37278 3728927 p2 (TA)5 10 43035 4304428 p1 (T)12 12 44264 4427529 c (T)10(A)12 88 45170 4525730 p1 (T)10 10 45494 4550331 p2 (TA)5 10 47486 4749532 p1 (T)10 10 49592 4960133 p3 (TTA)5 15 52259 5227334 p3 (TTA)4 12 52907 5291835 c (T)10(AAAT)3 75 55808 5588236 p1 (A)10 10 56446 5645537 p1 (T)10 10 58624 5863338 p1 (T)10 10 61005 6101439 p4 (CAAA)3 12 65603 6561440 p1 (T)10 10 68620 6862941 p2 (TA)5 10 69618 6962742 p4 (AAAC)3 12 69738 6974943 p1 (T)16 16 70813 7082844 p1 (T)15 15 71352 7136645 p1 (T)12 12 72308 7231946 p1 (A)12 12 72471 7248247 p1 (T)12 12 73307 7331848 p1 (A)10 10 75860 7586949 p1 (T)10 10 79899 7990850 p1 (T)14 14 85933 8594651 p2 (AT)5 10 86572 8658152 p1 (A)10 10 113920 11392953 p1 (A)10 10 114748 11475754 p4 (TTGA)3 12 118559 11857055 p2 (AT)5 10 122198 12220756 p2 (AT)5 10 154156 15416557 p1 (A)14 14 154791 154804

Table S6. Distribution of SSRs present in the L. barbarum, L. chinense, and L. ruthenicum chloroplast

genomes.

Taxon

Genome Size

(bp)

AT

(%)

SSR type CDS

Mono

D

i Tri

Tetr

a Penta Hexa

Tota

l %a

No.

b %c

① 155,656

62.

2 35

1

0 2 11 0 0 58 51.1 6 10.3

② 155,745

62.

2 32 8 3 11 0 0 54 51.1 6 11.1

③ 154,869

62.

1 40 9 3 9 1 0 62 50.3 5 8.1

①L.barbarum; ②L.chinense; ③L.ruthenicumCDS: protein-coding regions. a Percentage were calculated according to the total length of the CDS divided by the

genome size. b Total number of SSRs identified in the CDS. c Percentage were calculated according to the total

number of SSRs in the CDS divided by the total number of SSRs in the genome.

Table S7. DNA quality of three Lycium species.

Sample Nucleic acid conc (ng/uL) A260/280 A260/230L. chinense 43.3 1.78 1.79

L. barbarum

80.2 1.78 2.11

L. ruthenicum

169.8 1.73 1.77

Table S8. Primer sequences at the boundaries between single copy and IR regions.

Species Regions Forward/Reverse Primer sequence (5' to 3')

L.barbarumLSC-IRa

F TGCTCGATAGGGCATGAGTTCR ACAAGTGGGGAATGTTGGGG

IRa-SSCF AACAAGTGGTTTTGTTGGTTGGTR AGGATGCAGTTCTTTCAGTCAGT

SSC-IRbF TGTCCAGTTCATTAGGGAGCGR ACAAGTGGTTTTGTTGGTTGGT

IRb-LSCF GTCGGACAAGTGGGGAATGT

RGGAGGTTTTGAAAAGAAAGGAGCA

L.chinenseLSC-IRa

F TAGGAACGTCCGCGAATTTGAR CAAGTGGGGAATGTTGGGGT


SSC-IRbF TGTCCAGTTCATTAGGGAGCGR ACAAGTGGTTTTGTTGGTTGGT

IRb-LSCF GCAAAAGAGGGGAAATCGGC

R GGAGAGGGTATTTGCCTGCAT

L.ruthenicumLSC-IRa

F TAGGAACGTCCGCGAATTTGAR CAAGTGGGGAATGTTGGGGT


SSC-IRbF GTCCAGTTCATTAGGGAGCGTR ACAAGTGGTTTTGTTGGTTGGTT

IRb-LSC

F GCAAAAGAGGGGAAATCGGC

RGTTCTATCAAGAAGGTGCTATTGCT

Table S9. GenBank accession numbers of dicots with complete chloroplast genome sequences used

for phylogenetic analyses.

GenBank acc. Species GenBank acc. Species

NC_030056 Acnistus arborescens x Iochroma cyaneum NC_030282 Scopolia parvifloraKU568472 Acnistus arborescens NC_026879 Solanum habrochaitesNC_004561 Atropa belladonna KY887588 Solanum lycopersicumKR078311 Capsicum annuum var. glabriusculum NC_035516 Cressa creticaKR078314 Capsicum baccatum var. baccatum NC_009765 Cuscuta gronoviiNC_018117 Datura stramonium EU189133 Cuscuta obtusifloraKP308151 Dunalia brachyacantha EU118126 Ipomoea purpureaNC_026563 Dunalia obovata KF242476 Ipomoea trifidaNC_030171 Eriolarynx fasciculata KJ953908 Oncinotis tenuilobaKF248009 Hyoscyamus niger KJ953909 Pentalinon luteumNC_026694 Saracha punctata KM067394 Panax ginsengKU310932 Iochroma cardenasianum KM489056 Solanum tuberosumNC_007500 Nicotiana sylvestris KX426465 Ilex latifoliaKU199713 Nicotiana tabacumKP295964 Physalis peruviana

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