Supplementary table 1 Primer sequences used in RAPD and RT-PCR

Primer Type / Primer Name / Primer Sequence (5’ to 3’)
RAPD / OPC-15 / GACGGATCAG
RAPD / OPD-7 / TTGGCACGGG
RAPD / OPD-8 / GTGTGCCCCA
RAPD / OPD-11 / AGCGCCATTG
RAPD / OPD-13 / GGGGTGACGA
RAPD / OPD-20 / ACCCGGTCAC
RAPD / OPE-01 / CGCAAGGTCC
RAPD / OPE-07 / AGATGCAGCC
RAPD / OPE-09 / CTTCACCCGA
RAPD / OPE-12 / TTATCGCCCC
RAPD / OPE-15 / ACGCACAACC
RAPD / OPE-16 / GGTGACTGTG
RT-PCR / ADS / F: CGGGATCCATGTCACTTACAGAAG
R: CGGAATTCTCATATACTCATAGGA
RT-PCR / CYP71AV1 / F: GGGGTTAGGGATTTAGCCAGAA
R: AATTGCCTCCAGTACTCACCATAA
RT-PCR / DBR2 / F: ATAAATGACAGAACGGACGAA
R: GTCAAGAGAAGGGTAATCCGT
RT-PCR / ALDH1 / F: GGACTTGCCTCAGGTGTAT
R: GTGCCTCTAATCCTTGTTC
RT-PCR / RPS9 / F: GCGTTTGGATGCTGACTTGA
R: GGCGCTCAAGGAAGTTCTCTAC
RT-Q-PCR / ADS / F: ATACAACGGGCACTAAAGCAACC
R: GAAAACTCTAGCCCGGGAATACTG
RT-Q-PCR / CYP71AV1 / F: GGGGTTAGGGATTTAGCCAGAA
R: AATTGCCTCCAGTACTCACCATAA
RT-Q-PCR / 18SrRNA / F: TCCGCCGGCACCTTATGAGAAATC
R: CTAAGAACGGCCATGCACCACCAC

Supplementary table 2Morphological descriptors of wild type and 13 induced salt tolerant somaclonal variantsof Artemisia annua

Relevant strain/
Genotype / Important features of the plant at the different stage of plants. / Origin/
Reference
Micro propagation stage (In vitro) / Seedling stage / Young plant stage
Control / Compact habit, long petiole, leaves very dark green in colour (139B), large no. of branches, lacerate margin / Cotyledon ovate in shape, first leaf elliptical shape, Sessile, broad leaf lamina, serrate margin in upper portion of leaf, alternate leaf. / Lacerate margin of leaves, long petiole, dark green in colour (137A), early branching. / (Kumar et al., 1999)
ASV1 / Compact habit, medium size of internode, less no. of branches, compact leaf lamina, leaf green in colour (137A). / Cotyledon ovate in shape, first leaf obovate in shape, Sessile, alternate leaf, slightly lacerate margin. / Whorled leaf separated from base, large size of petiole, slightly dissected leaf, small leaf lamina, green in colour (137A). / γ rays mutagenesis
ASV2 / Compact habit, less no. of branch, thick petiolated leaf, serration in entire margin of leaf, green in colour (137B). / Cotyledon obovate in shape, first leaf elliptic in shape, texture waxy, leaf margin smooth. / Small size thick petiole, broad leaf lamina, alternate branches, serration in upper half margin of leaf, green in colour (137B). / γ rays mutagenesis
ASV3 / Thick nodal & intermodal region, intermodal length medium size, leaf lamina narrow, leaf margin lacerate, green in colour (137A). / Cotyledon obovate in shape, first leaf have spatulate in shape with long thick petiole, serration in middle part of leaf, green in colour (137A). / First leaf spatulate in shape, second have serration, thick lamina in margin, slightly lobed in leaf, small internode, yellowish green in colour (147B). / γ rays mutagenesis
ASV4 / Thick stem, alternate branches, long petiole, small leaf lamina, green in color (137B) / Cotyledon spatulate, long petiole, first leaf sessile, leaf pubiscent, leaf hairy, green in colour (137B). / Elliptic shape of leaf, small internode, long petiole, tricate in position of leaf, serration in upper margin of leaf, green in colour (137B), deeply lobed margin. / γ rays mutagenesis
ASV5 / Thick nodal region, long internode, alternate leaf with broad leaf lamina, light green in colour (137A). / Cotyledon lanceolate, elliptic in shape, petiole thick, broad leaf lamina with slight serration. / Dwarf structure, very long petiole, green in colour(137B), leaf lamina slightly dissected / γ rays mutagenesis
ASV6 / Stem thick with long internode, leaf whorled, light green in colour(137B), curved leaf lamina. / Cotyledon single, first leaf like cotyledon, small in size, second leaf highly lobed. / Cotyledon abnormal in shape having long petiole, cup shaped lamina, first two leaf ovate in shape just like cotyledon, Cotyledon persistent for long time, small leaf with 4-5 lobes & short petiole. / γ rays mutagenesis
ASV7 / Leaves are alternate, petiolate, green in colour with small lamina (147B). / Cotyledon elliptic in shape with mucronate apex, green in colour, first leaf small in size & less lobed. / Compact habit, broad leaf lamina, long petiole, dark green in colour. / γ rays mutagenesis
ASV8 / Thin stem with whorled leaves, leaves are petiolated & small leaf lamina, green color (137B) / Cotyledon obovate in shape having serration , first leaf lobed with short petiole, elliptic in shape, green in colour (137B) / Dwarf plant, slow growing, medium size petiole with highly dissected narrow lamina, green in colour (137B) / γ rays mutagenesis
ASV9 / Compact, bushy with short internodes, large no. of branches, leaf small in size with thick lamina, yellowish green color (147B) / Cotyledon spatulate with serrated margin, first leaf having thick petiole, broad leaf lamina, light green in colour (137B). / Dwarf, slow growing plant with thick petiole, small sized highly lobed leaves, no. of lobes are less, green in colour (137B). / γ rays mutagenesis
ASV10 / Highly compact habit with very small size of internode, large no. of branches with whorled leaf, compactly arranged, yellowish green color (147B). / Cotyledon spatulate in shape with long petiole & serrated margin, first leaf having thick broad leaf lamina, green in colour (137B). / Fast growing plant with large no. of leaves, early branching, broad leaf lamina, leaf lamina highly dissected, green in colour (137B). / γ rays mutagenesis
ASV11 / Highly compact habit with single internode, whorled leaves, slow growing, green in colour (137B). / Cotyledon obovate in shape with entire margin, first leaf abnormal in size, lacerate margin, green color (137B) / Compact habit, slow growing, long petiole, green in colour (137B) with broad leaf lamina. / γ rays mutagenesis
ASV12 / Stem highly dark green in color (139B), long internode, leaf with short petiole & thick lamina, very large leaves / Cotyledon obovate in shape first leaves small in size with thick lamina. / Plant slow growing with compact lamina, long petiole, green in colour (137B). / γ rays mutagenesis
ASV13 / Thick stem with small size, tiny leaves, green in colour (137B) / Cotyledon elliptic in shape, first leaf abnormal in shape with lacerate margin, green colour (137B) / First two leaf lanciolate in shape, petiolated, lacerate margin, green in color (137B) / γ rays mutagenesis

Given in parenthesis are the color codes of Royal Horticulture Society (RHS): 137A, 137B, 139A, 139B=green group; 146A, 147 A, 147B= yellow green group

Supplementary table 3Number of polymorphic bands produced by different primers in wild type and 13 induced salt tolerant somaclonal variants of Artemisia annua

Primer Name / No. of amplified products / No. of polymorphic bands / Percent Polymorphism (%)
OPC-15 / 12 / 2 / 18.18
OPD-7 / 5 / 3 / 60.00
OPD-8 / 14 / 14 / 100.00
OPD-11 / 10 / 3 / 30.00
OPD-13 / 15 / 9 / 60.00
OPD-20 / 11 / 4 / 36.36
OPE-01 / 7 / 3 / 42.85
OPE-07 / 18 / 9 / 50.00
OPE-09 / 3 / 0 / 00.00
OPE-12 / 8 / 4 / 50.00
OPE-15 / 5 / 0 / 00.00
OPE-16 / 15 / 3 / 20.00
Total number of amplified bands / 123 / 54

Supplementary table 4Similarity matrices of the wild type and 13 induced salt tolerant somaclonal variant of Artemisia annua deduced from band patterns of PCR amplified genomic DNAs using agarose gel electrophoresis

Control / ASV1 / ASV2 / ASV3 / ASV4 / ASV5 / ASV6 / ASV7 / ASV8 / ASV9 / ASV10 / ASV11 / ASV12
ASV1 / -0.01
ASV2 / 0.11 / 0.49**
ASV3 / 0.16* / 0.20* / 0.12
ASV4 / 0.14 / 0.10 / 0.06 / 0.41**
ASV5 / 0.05 / 0.19* / 0.13 / 0.45** / 0.29**
ASV6 / 0.06 / 0.13 / 0.12 / 0.29** / 0.32** / 0.28**
ASV7 / -0.01 / 0.95** / 0.44** / 0.18* / 0.07 / 0.13 / 0.08
ASV8 / 0.10 / 0.04 / 0.07 / 0.04 / 0.02 / 0.03 / -0.06 / -0.01
ASV9 / 0.05 / 0.09 / 0.09 / 0.06 / 0.09 / 0.16 / 0.10 / 0.04 / 0.26*
ASV10 / 0.08 / -0.01 / 0.12 / 0.13 / 0.10 / 0.19* / 0.15 / -0.07 / 0.15 / 0.25**
ASV11 / 0.00 / 0.09 / 0.05 / 0.23** / 0.15 / 0.30** / 0.15 / 0.03 / 0.17* / 0.17* / 0.00
ASV12 / -0.12 / -0.09 / 0.08 / 0.23** / 0.19* / 0.07 / 0.04 / -0.07 / -0.08 / 0.01 / -0.05 / 0.12
ASV13 / 0.15 / 0.14 / 0.04 / 0.15 / 0.10 / 0.25** / 0.19* / 0.09 / 0.03 / 0.15 / 0.29** / 0.07 / 0.02

Supplementary table 5Variation in 16 metric traits parameters among wild type and 13 induced salt tolerant somaclonal variants in Artemisia annua

Sl. No. / Trait designation / Code / Mean / Range
Minimum / Maximum
1 / Root Length (cm plant-1) / RL / 21.31 / 9 / 29.05
2 / Plant Height (cm plant-1) / PH / 198.27 / 65.7 / 275
3 / No. of Branches (plant-1) / NOB / 36.30 / 18.67 / 43.65
4 / No. of Leaves(plant-1) / NOL / 649.53 / 482.92 / 1107
5 / Stem Fresh Weight (g plant-1) / SFW / 193.33 / 44.05 / 258.06
6 / Leaf Fresh Weight (g plant-1) / LFW / 155.37 / 44.23 / 286.43
7 / Total Fresh Weight (g plant-1) / TFW / 350.52 / 89 / 640
8 / Stem Dry Weight (g plant-1) / SDW / 102.47 / 13 / 164
9 / Leaf Dry Weight (g plant-1) / LDW / 60.32 / 13.01 / 109.56
10 / Total Dry Weight (g plant-1) / TDW / 166.35 / 24 / 296
11 / Leaf Artemisinin Content (% dw) / LAC / 0.396 / 0.36 / 0.526
12 / Stem Artemisinin Content (% dw) / SAC / 0.0918 / 0.03 / 0.091
13 / Total Artemisinin Yield (Kg ha-1) / TAY / 12.22 / 2.187 / 29.08
14 / Leaf Artemisinin Yield (Kg ha-1) / LAY / 9.9764 / 1.75 / 23.1
15 / Stem Dry Yield (Kg ha-1) / SDY / 4260.05 / 618 / 7480
16 / Leaf Dry Yield (Kg ha-1) / LDY / 2416.25 / 518 / 4395

Supplementary table 6Co-efficient of correlations (r) between pairs of Artemisinin yield related characters in wild type and 13 induced salt tolerant somaclonal variants of Artemisia annua

RL / PH / NOB / NOL / SFW / LFW / TFW / SDW / LDW / TDW / LAC / SAC / TAY / LAY / SDY
PH / 0.92** / 1
NOB / 0.95** / 0.92** / 1
NOL / 0.48** / 0.57** / 0.67** / 1
SFW / 0.92** / 0.95** / 0.97** / 0.73** / 1
LFW / 0.55** / 0.61** / 0.73** / 0.98** / 0.78** / 1
TFW / 0.77** / 0.80** / 0.88** / 0.90** / 0.93** / 0.94** / 1
SDW / 0.87** / 0.98** / 0.90** / 0.64** / 0.95** / 0.67** / 0.83** / 1
LDW / 0.86** / 0.93** / 0.94** / 0.77** / 0.97** / 0.82** / 0.92** / 0.94** / 1
TDW / 0.78** / 0.90** / 0.81** / 0.62** / 0.88** / 0.65** / 0.78** / 0.92** / 0.87** / 1
LAC / 0.15 / 0.08 / 0.33* / 0.77** / 0.34* / 0.77** / 0.63** / 0.14 / 0.36* / 0.12 / 1
SAC / -0.45** / -0.47** / -0.46** / -0.13 / -0.47** / -0.22 / -0.36** / -0.41* / -0.38* / -0.4** / 0.038 / 1
TAY / 0.69** / 0.76** / 0.83** / 0.94** / 0.88** / 0.96** / 0.97** / 0.82** / 0.91** / 0.78** / 0.63** / -0.26 / 1
LAY / 0.72** / 0.78** / 0.85** / 0.93** / 0.90** / 0.96** / 0.98** / 0.83** / 0.93** / 0.78** / 0.63** / -0.27 / 0.99** / 1
SDY / 0.87** / 0.98** / 0.90** / 0.64** / 0.95** / 0.67** / 0.83** / 0.99** / 0.95** / 0.92** / 0.13 / -0.42** / 0.82** / 0.83** / 1
LDY / 0.86** / 0.93** / 0.94** / 0.76** / 0.97** / 0.81** / 0.92** / 0.94** / 0.99** / 0.87** / 0.35* / -386* / 0.91** / 0.92** / 0.95**

** Correlation is significant at the 0.01 level (2-tailed) * Correlation is significant at the 0.05 level (2-tailed) RL (Root Length), PH (Plant Height), NOB (No. of Branches), NOL (No. of leaves), SFW (Stem FW), LFW (Leaf FW), TFW (Total FW), SDW (Stem DW), LDW (Leaf DW), TDW (Total DW), LAC (Leaf Artemisinin Content), SAC (Stem artemisinin content), TAY (Total artemisinin yield), LAY (Leaf artemisinin yield), SDY (Stem dry yield), LDY (Leaf dry yield)

Supplementary table 7Eigen vector contributions by principle component analysis estimated in wild type (control) and in a collection of 13 induced salt tolerant somaclonal variants of Artemisia annua

Variable / PC1 / PC2
Control / 1.5 / -1.7
ASV1 / 7.6 / 0.5
ASV2 / 6.1 / -0.8
ASV3 / 0.9 / 1.0
ASV4 / 1.6 / -0.2
ASV5 / 1.4 / 0.0
ASV6 / 2.3 / -0.6
ASV7 / 7.6 / 0.8
ASV8 / 3.8 / -5.6
ASV9 / 4.2 / -4.9
ASV10 / 1.4 / -3.5
ASV11 / 2.2 / -2.1
ASV12 / -0.6 / 0.6
ASV13 / 3.2 / -2.5

Supplementaytable 8Means and standard errors of the 16 morphological traits evaluated among 6 clusters formed by wild type and 13 induced salt tolerant somaclonal variants of Artemisia annua

Trait / Cluster I (ASV3, ASV4, ASV5, ASV6) / Cluster II (ASV1, ASV7, ASV2) / Cluster III (ASV12) / Cluster IV(ASV13, ASV11) / Cluster V(ASV8, ASV9) / Cluster VI(Control, ASV10)
RL(cm plant-1) / 9.87
±0.97 / 17.55
±1.18 / 11.78
±0.97 / 23.33
±1.24 / 13.11
±1.24 / 10.12
±1.7
PH(cm plant-1) / 67.78
±3.47 / 230.05
±4.99 / 292.00
±8.89 / 157.67
±8.411 / 288.67
±8.41 / 139.33
±7.88
NOB(plant-1) / 24.33
±1.86 / 53.00
±2.31 / 75.00
±3.21 / 42.12
±4.32 / 40.12
±2.36 / 45.01
±3.05
NOL(Plant-1) / 325.00
±13.23 / 342.01
±10.25 / 597.33
±14.95 / 421.05
±8.95 / 400
±8.95 / 401
±10.97
SFW(g plant-1) / 98.11
±2.76 / 46.16
±1.96 / 148.17
±3.78 / 121.31
±6.15 / 126.89
±6.15 / 148.17
±3.28
LFW(g plant-1) / 44.95
±2.64 / 35.38
±2.82 / 327.17
±4.56 / 90.27
±4.14 / 112.50
±4.41 / 158
±4.65
TFW(g plant-1) / 245.58
±1.65 / 71.54
±2.42 / 527.49
±11.20 / 61.67
±3.14 / 140
±3.14 / 91.11
±0.85
SDW(g plant-1) / 61.67
±2.42 / 175.53
±3.07 / 140.13
±3.14 / 154.62
±3.90 / 157.92
±3.50 / 15.48
±1.63
LDW(g plant-1) / 45.65
±1.11 / 41.86
±1.53 / 109.95
±2.17 / 59.59
±1.07 / 72.42
±1.07 / 13.02
±0.57
TDW(g plant-1) / 76.03
±4.04 / 90.43
±2.74 / 113.41
±1.68 / 106.20
±6.4 / 76.03
±6.4 / 28.49
±1.31
LAC (% dw) / 0.305
±0.004 / 0.523
±0.003 / 0.908
±0.003 / 0.493
±0.001 / 0.496
±0.001 / 0.316
±0.001
SAC (% dw) / 0.032
±0.001 / 0.041
±0.001 / 0.80
±0.001 / 0.062
±0.001 / 0.056
±0.001 / 0.073
±0.001
TAY (Kg ha-1) / 8.643
±0.193 / 21.057
±0.512 / 29.96
±0.609 / 21.463
±0.292 / 16.427
±0.292 / 11.047
±0.332
LAY(Kg ha-1) / 6.943
±0.193 / 18.28
±0.263 / 23.21
±0.532 / 15.42
±0.15 / 7.43
±0.15 / 16.129
±0.468
SDY(Kg ha-1) / 52.7
±23.41 / 6156.7
±140.05 / 7481
±123.7 / 1982
±112.12 / 1661
±112.12 / 619.0
±64.97
LDY(Kg ha-1) / 1674
±61.35 / 925.0
±28.58 / 3573
±109.17 / 1379
±26.85 / 1634
±26.85 / 520.7
±22.66