Developing and Testing Molecular Markers in Cannabis sativa (Hemp) for Their Use in Variety and Dioecy Assessments
Abstract
:1. Introduction
2. Results and Discussion
2.1. Overall Genetic Diversity
2.2. Population Structure of Cannabis Germplasms and Cluster Analysis
2.3. Sex Determination and Linkage Disequilibrium
3. Materials and Methods
3.1. Plant Materials of Cannabis
3.2. Analysis of the SSR Marker Loci
3.3. Molecular Data Analysis
3.4. Prediction of Plant Sex through the SCAR119 Marker and Linkage Disequilibrium Analysis
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Locus | General Statistics | H-Statistics | F-Statistics | Nm | ||||||
---|---|---|---|---|---|---|---|---|---|---|
Na | pi | PIC | Ho | He | Ha | Fis | Fit | Fst | ||
SSR_6–3 | 7 | 0.54 | 0.57 | 0.50 | 0.62 | 0.25 | 0.00 | 0.22 | 0.22 | 0.86 |
SSR_2–2 | 7 | 0.55 | 0.58 | 0.42 | 0.63 | 0.21 | 0.16 | 0.37 | 0.25 | 0.60 |
SSR_X-1 | 28 | 0.13 | 0.91 | 0.53 | 0.92 | 0.25 | 0.42 | 0.46 | 0.07 | 1.40 |
SSR_4–2 | 11 | 0.43 | 0.70 | 0.22 | 0.74 | 0.10 | 0.73 | 0.76 | 0.10 | 0.69 |
SSR_2–3 | 24 | 0.25 | 0.88 | 0.70 | 0.89 | 0.34 | 0.18 | 0.24 | 0.08 | 1.69 |
SSR_7–3 | 17 | 0.21 | 0.88 | 0.83 | 0.89 | 0.41 | 0.03 | 0.10 | 0.07 | 1.67 |
SSR_3–3 | 23 | 0.20 | 0.90 | 0.70 | 0.91 | 0.34 | 0.19 | 0.26 | 0.09 | 1.47 |
SSR_2–1 | 7 | 0.77 | 0.35 | 0.07 | 0.38 | 0.03 | 0.74 | 0.84 | 0.37 | 0.23 |
SSR_4–1 | 11 | 0.70 | 0.47 | 0.20 | 0.50 | 0.09 | 0.68 | 0.72 | 0.13 | 0.42 |
SSR_8–2 | 20 | 0.17 | 0.90 | 0.84 | 0.91 | 0.39 | 0.07 | 0.16 | 0.10 | 1.33 |
SSR_5–2 | 21 | 0.14 | 0.91 | 0.32 | 0.92 | 0.13 | 0.67 | 0.73 | 0.17 | 0.37 |
SSR_6–1 | 14 | 0.18 | 0.86 | 0.35 | 0.88 | 0.14 | 0.64 | 0.69 | 0.14 | 0.78 |
SSR_X-3 | 25 | 0.13 | 0.92 | 0.70 | 0.93 | 0.29 | 0.31 | 0.37 | 0.10 | 0.87 |
SSR_1–4 | 8 | 0.62 | 0.56 | 0.57 | 0.59 | 0.26 | 0.16 | 0.21 | 0.06 | 2.78 |
SSR_3–1 | 15 | 0.16 | 0.88 | 0.52 | 0.90 | 0.22 | 0.45 | 0.53 | 0.14 | 1.05 |
SSR_8–4 | 17 | 0.16 | 0.88 | 0.41 | 0.90 | 0.18 | 0.56 | 0.61 | 0.12 | 0.78 |
SSR_9–4 | 17 | 0.23 | 0.88 | 0.63 | 0.89 | 0.27 | 0.35 | 0.41 | 0.09 | 1.24 |
SSR_1–1 | 18 | 0.23 | 0.87 | 0.36 | 0.88 | 0.14 | 0.63 | 0.69 | 0.15 | 0.63 |
SSR_5–5 | 8 | 0.70 | 0.46 | 0.08 | 0.49 | 0.03 | 0.86 | 0.89 | 0.21 | 0.51 |
SSR_6–4 | 3 | 0.67 | 0.40 | 0.38 | 0.47 | 0.17 | 0.29 | 0.42 | 0.17 | 1.05 |
Mean | 15.05 | 0.36 | 0.74 | 0.47 | 0.76 | 0.21 | 0.41 | 0.48 | 0.14 | 1.02 |
St. Dev. | 7.12 | 0.23 | 0.20 | 0.23 | 0.19 | 0.11 | 0.27 | 0.24 | 0.08 | 0.13 |
Variety | N | General Statistics | H-Statistics | F-Statistics | Nm | |||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
P | I | SM (%) | GS (%) | No | Ne | Ho | He | Ha | Fis | Fit | Fst | |||
ITA1 | 10 | 0.90 | 1.19 ± 0.13 | 65.68 ± 4.42 | 88.68 ± 1.71 | 4.90 ± 0.56 | 3.11 ± 0.32 | 0.59 ± 0.05 | 0.62 ± 0.06 | 0.26 ± 0.16 | 0.13 | 0.12 | 0.56 | 0.19 |
ITA2 | 9 | 0.95 | 1.15 ± 0.12 | 64.21 ± 4.80 | 89.16 ± 1.62 | 4.40 ± 0.48 | 3.15 ± 0.36 | 0.58 ± 0.05 | 0.63 ± 0.06 | 0.20 ± 0.17 | 0.27 | 0.36 | 0.68 | 0.12 |
ITA3 | 10 | 1.00 | 1.49 ± 0.09 | 67.96 ± 3.76 | 86.62 ± 1.55 | 6.15 ± 0.47 | 4.00 ± 0.36 | 0.70 ± 0.03 | 0.74 ± 0.03 | 0.25 ± 0.14 | 0.26 | 0.29 | 0.64 | 0.14 |
HUN1 | 11 | 1.00 | 1.15 ± 0.11 | 67.50 ± 5.67 | 89.96 ± 1.67 | 4.60 ± 0.46 | 3.03 ± 0.32 | 0.59 ± 0.05 | 0.62 ± 0.05 | 0.19 ± 0.13 | 0.31 | 0.40 | 0.70 | 0.11 |
HUN2 | 10 | 1.00 | 1.35 ± 0.13 | 67.36 ± 3.31 | 87.09 ± 1.47 | 5.75 ± 0.63 | 3.80 ± 0.49 | 0.64 ± 0.04 | 0.68 ± 0.05 | 0.22 ± 0.13 | 0.24 | 0.35 | 0.68 | 0.12 |
FIN | 11 | 0.85 | 1.41 ± 0.17 | 69.76 ± 2.97 | 87.54 ± 1.27 | 6.15 ± 0.74 | 4.43 ± 0.55 | 0.63 ± 0.07 | 0.66 ± 0.07 | 0.22 ± 0.17 | 0.34 | 0.32 | 0.66 | 0.13 |
NED | 13 | 0.90 | 1.15 ± 0.14 | 65.67 ± 5.88 | 89.37 ± 1.77 | 4.60 ± 0.50 | 3.29 ± 0.40 | 0.57 ± 0.06 | 0.59 ± 0.06 | 0.18 ± 0.15 | 0.35 | 0.38 | 0.69 | 0.11 |
POL | 8 | 1.00 | 1.04 ± 0.12 | 64.16 ± 4.91 | 89.91 ± 1.34 | 3.95 ± 0.41 | 2.77 ± 0.32 | 0.54 ± 0.05 | 0.58 ± 0.05 | 0.21 ± 0.16 | 0.22 | 0.26 | 0.63 | 0.15 |
FRA1 | 10 | 0.95 | 1.22 ± 0.14 | 63.57 ± 6.13 | 88.27 ± 2.15 | 4.70 ± 0.51 | 3.57 ± 0.42 | 0.60 ± 0.06 | 0.64 ± 0.06 | 0.18 ± 0.16 | 0.31 | 0.45 | 0.73 | 0.09 |
FRA2 | 9 | 1.00 | 1.39 ± 0.12 | 63.36 ± 4.30 | 86.26 ± 1.81 | 5.45 ± 0.56 | 4.02 ± 0.50 | 0.67 ± 0.04 | 0.72 ± 0.04 | 0.21 ± 0.13 | 0.32 | 0.40 | 0.70 | 0.11 |
FRA3 | 3 | 0.65 | 0.56 ± 0.11 | 49.86 ± 9.43 | 91.08 ± 2.13 | 2.10 ± 0.25 | 1.89 ± 0.21 | 0.34 ± 0.06 | 0.48 ± 0.09 | 0.17 ± 0.16 | 0.02 | 0.09 | 0.54 | 0.21 |
SCAR | SSR_ 1–1 | SSR_ 1–4 | SSR_ 2–1 | SSR_ 2–2 | SSR_ 2–3 | SSR_ 3–1 | SSR_ 3–3 | SSR_ 4–1 | SSR_ 4–2 | SSR_ 5–2 | SSR_ 5–5 | SSR_ 6–1 | SSR_ 6–3 | SSR_ 6–4 | SSR_ 7–3 | SSR_ 8–2 | SSR_ 8–4 | SSR_ 9–4 | SSR_ X-1 | SSR_ X-3 | |
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
SCAR | × | ** | ** | ||||||||||||||||||
SSR_1–1 | 0.025 | × | ** | ** | ** | ** | ** | ** | ** | ||||||||||||
SSR_1–4 | 0.533 | 0.167 | × | ||||||||||||||||||
SSR_2–1 | 0.008 | 0.002 | 0.031 | × | ** | ** | ** | ** | ** | ** | *** | ** | ** | ** | ** | *** | *** | ||||
SSR_2–2 | 0.115 | 0.062 | 0.097 | 0.003 | × | ** | ** | ** | ** | ||||||||||||
SSR_2–3 | 0.900 | 0.032 | 0.871 | 0.002 | 0.410 | × | |||||||||||||||
SSR_3–1 | 0.092 | 0.005 | 0.053 | 0.003 | 0.029 | 0.132 | × | *** | ** | *** | *** | *** | ** | ** | *** | ** | |||||
SSR_3–3 | 0.103 | 0.036 | 0.254 | 0.051 | 0.121 | 0.617 | 0.044 | × | ** | ** | ** | ** | |||||||||
SSR_4–1 | 0.014 | 0.019 | 0.215 | 0.008 | 0.005 | 0.056 | 0.013 | 0.006 | × | *** | ** | ** | ** | ||||||||
SSR_4–2 | 0.014 | 0.005 | 0.015 | 0.002 | 0.002 | 0.194 | <0.001 | 0.002 | <0.001 | × | *** | *** | ** | ** | ** | *** | ** | ** | |||
SSR_5–2 | 0.058 | 0.013 | 0.227 | 0.005 | 0.021 | 0.238 | 0.009 | 0.030 | 0.016 | <0.001 | × | ** | ** | ** | |||||||
SSR_5–5 | 0.003 | 0.002 | 0.016 | <0.001 | 0.001 | 0.014 | <0.001 | 0.005 | 0.003 | <0.001 | 0.001 | × | ** | *** | ** | *** | ** | ** | *** | *** | ** |
SSR_6–1 | 0.028 | 0.048 | 0.026 | 0.004 | 0.004 | 0.082 | <0.001 | 0.046 | 0.042 | 0.004 | 0.011 | 0.002 | × | ** | ** | ** | ** | ||||
SSR_6–3 | 0.253 | 0.135 | 0.916 | 0.028 | 0.103 | 0.484 | 0.308 | 0.342 | 0.018 | 0.003 | 0.037 | <0.001 | 0.021 | × | |||||||
SSR_6–4 | 0.089 | 0.012 | 0.333 | 0.007 | 0.047 | 0.790 | 0.100 | 0.111 | 0.063 | 0.020 | 0.118 | 0.002 | 0.007 | 0.294 | × | ||||||
SSR_7–3 | 0.285 | 0.016 | 0.518 | 0.006 | 0.019 | 0.138 | <0.001 | 0.090 | 0.014 | 0.001 | 0.015 | <0.001 | 0.002 | 0.243 | 0.100 | × | ** | ||||
SSR_8–2 | 0.806 | 0.019 | 0.486 | 0.006 | 0.078 | 0.170 | 0.051 | 0.107 | 0.122 | 0.014 | 0.026 | 0.010 | 0.120 | 0.369 | 0.431 | 0.168 | × | ||||
SSR_8–4 | 0.060 | 0.003 | 0.058 | 0.011 | 0.078 | 0.042 | 0.001 | 0.019 | 0.015 | <0.001 | 0.007 | 0.009 | 0.032 | 0.113 | 0.069 | 0.040 | 0.085 | × | ** | ** | ** |
SSR_9–4 | 0.083 | 0.008 | 0.070 | <0.001 | 0.126 | 0.250 | 0.002 | 0.010 | 0.019 | 0.002 | 0.046 | <0.001 | 0.008 | 0.144 | 0.162 | 0.225 | 0.447 | 0.010 | × | ** | |
SSR_X-1 | 0.035 | 0.006 | 0.180 | <0.001 | 0.102 | 0.041 | <0.001 | 0.084 | 0.003 | 0.016 | 0.057 | <0.001 | 0.007 | 0.063 | 0.132 | 0.003 | 0.145 | 0.007 | 0.003 | × | |
SSR_X-3 | 0.205 | 0.034 | 0.529 | 0.018 | 0.160 | 0.380 | 0.007 | 0.024 | 0.003 | 0.003 | 0.004 | 0.001 | 0.015 | 0.197 | 0.350 | 0.051 | 0.209 | 0.004 | 0.184 | 0.015 | × |
Variety | Origin | N. of Samples | Sex Behavior of the Samples | Leaf | ||
---|---|---|---|---|---|---|
Dioecious (Male) | Dioecious (Female) | Monoecious | ||||
ITA1 | Italy | 10 | 7 | 3 | ||
ITA2 | Italy | 9 | 4 | 5 | ||
ITA3 | Italy | 10 | 2 | 8 | ||
HUN1 | Hungary | 11 | 6 | 5 | ||
HUN2 | Hungary | 10 | 5 | 5 | ||
FIN | Finland | 11 | 3 | 8 | ||
NED | Netherlands | 13 | 13 | |||
POL | Poland | 8 | 8 | |||
FRA1 | France | 10 | 10 | |||
FRA2 | France | 9 | 9 | |||
FRA3 | France | 3 | 3 |
Locus Name | Start | End | Expected Size | Multiplex | Fluo Dye | Ta (°C) | Motif | Forward Primer | Reverse Primer |
---|---|---|---|---|---|---|---|---|---|
SSR_6–3 | 35,062,092 | 35,062,261 | 180–200 | 1 | M13 | 55 | (AAT)10 | ATCTCATTTTCCGTACCTGTT | CTAATTCTCAACTTAACCGCG |
SSR_2–2 | 27,019,093 | 27,019,345 | 250–270 | 1 | M13 | 55 | (TGA)12 | TAGTAGTAGTAGTGCCTGAGG | ACCTTAACAACACCACAACTA |
SSR_X-1 | 12,090,959 | 12,091,352 | 390–450 | 1 | M13 | 55 | (TC)40 | TTGTCAAGGGAGCTTAGTTAG | ATGTGTATTTCTCGCCTGTTA |
SSR_4–2 | 38,738,240 | 38,738,472 | 230–260 | 1 | PAN1 | 55 | (AT)17 | CAGAGTTTGGTCCTTTTCAAA | CACGGATTTTAAGCATTGGAT |
SSR_2–3 | 49,240,375 | 49,240,744 | 350–410 | 1 | PAN1 | 55 | (GA)22 | CTCCCTGCCATTAGACAAATA | CCAGGAGGTAATTTTCTGCTA |
SSR_7–3 | 51,776,452 | 51,776,692 | 230–280 | 1 | PAN2 | 55 | (CT)22 | ACTGTGAACTGTCCTTTTACA | AACAACCTGAAATCCGAAAAG |
SSR_3–3 | 59,258,629 | 59,258,880 | 250–300 | 1 | PAN3 | 55 | (AG)21 | CAAAGAAAGCAGGCATTAGTT | CTCTCTGTGAATGTGATCTGT |
SSR_2–1 | 15,695,145 | 15,695,388 | 240–260 | 2 | M13 | 55 | (AAT)11 | GGCAGGAAAAATCTCAAACAT | ACATTGGAATTAGACAGAGCA |
SSR_4–1 | 3,414,697 | 3,414,947 | 230–270 | 2 | PAN1 | 55 | (ATA)21 | GTTGGTTATGTGTTAGGGTCT | GTTATGGACAAACAATGCATG |
SSR_8–2 | 13,924,026 | 13,924,199 | 180–220 | 2 | PAN2 | 55 | (CT)21 | CATCACACCAGGTACCAATAT | CATGAAACAACGTTGGGTTAT |
SSR_5–2 | 34,558,385 | 34,558,643 | 250–300 | 2 | PAN2 | 55 | (CT)32 | TGGCTGAAAGTAAGAAAAGAC | TTATCGCTCAAAACACTCAAC |
SSR_6–1 | 3,764,859 | 3,765,058 | 200–270 | 2 | PAN3 | 55 | (AT)17 | ACTTCACATGAGATTGAGAACA | TCCTTTGGATTCATTAAGTTGT |
SSR_X-3 | 71,305,129 | 71,305,410 | 280–350 | 2 | PAN3 | 55 | (TC)41 | ACAGTAGTTTTCAGGGTTGAA | TCACACCAATATCTATCAGCC |
SSR_1–4 | 86,039,144 | 86,039,328 | 180–220 | 3 | M13 | 55 | (TTA)17 | TCAAGTTACGTAATCCCCAAA | CCTAAGCACAAGGTTAAATCAT |
SSR_3–1 | 12,247,530 | 12,247,829 | 300–340 | 3 | M13 | 55 | (TC)32 | TGATTTTGCGACCCTTTTATG | CTTTTGCAGGTACATCCAAAA |
SSR_8–4 | 50,925,135 | 50,925,396 | 280–330 | 3 | PAN1 | 55 | (TC)22 | TATGCATCCATTGTACCTGTT | TAATGTTTGTGTGTGTGCAAA |
SSR_9–4 | 58,895,568 | 58,895,670 | 110–150 | 4 | PAN1 | 57 | (CT)16 | TTTCCTGCTCACCTTAAACC | AACCTATATTGAGACGAACCG |
SSR_1–1 | 12,756,851 | 12,757,030 | 180–220 | 4 | PAN1 | 57 | (TC)33 | AAACTGACAGCTTAAGCATTC | TGGGCATGTACTCTATCACTA |
SSR_5–5 | 82,565,436 | 82,565,719 | 270–290 | 4 | PAN2 | 57 | (GA)18 | AGAGGAAGGAAAGAGAGCTAT | CACGAGGGAGCCTTATTAATA |
SSR_6–4 | 63,517,285 | 63,517,456 | 170–180 | 4 | PAN3 | 57 | (CT)30 | ACGAGACTTTACAGAGAACAA | AGATAGGGAAGAACACAACAC |
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Borin, M.; Palumbo, F.; Vannozzi, A.; Scariolo, F.; Sacilotto, G.B.; Gazzola, M.; Barcaccia, G. Developing and Testing Molecular Markers in Cannabis sativa (Hemp) for Their Use in Variety and Dioecy Assessments. Plants 2021, 10, 2174. https://doi.org/10.3390/plants10102174
Borin M, Palumbo F, Vannozzi A, Scariolo F, Sacilotto GB, Gazzola M, Barcaccia G. Developing and Testing Molecular Markers in Cannabis sativa (Hemp) for Their Use in Variety and Dioecy Assessments. Plants. 2021; 10(10):2174. https://doi.org/10.3390/plants10102174
Chicago/Turabian StyleBorin, Marcello, Fabio Palumbo, Alessandro Vannozzi, Francesco Scariolo, Gio Batta Sacilotto, Marco Gazzola, and Gianni Barcaccia. 2021. "Developing and Testing Molecular Markers in Cannabis sativa (Hemp) for Their Use in Variety and Dioecy Assessments" Plants 10, no. 10: 2174. https://doi.org/10.3390/plants10102174