Induced mutations contribute to genetic variability mainly by increasing DNA polymorphism [
However, molecular characterization of mutation events induced by irradiation treatments in
The seeds of
Then, the seeds were irradiated with different doses (20, 40, 60, 80, 100, 120, and 140 Kr) of 60Co-
Immediately after radiation, 200 treated seeds were sown at a depth of 1 cm with a mixed medium of river sand, red soil, and farm yard manure at the ratio of 3 : 2 : 1 in plastic pots (23 × 27 cm, 6 cm in height). After a month of germination, the number of germinated seeds was counted and expressed as percentage of the total number of seeds planted. Ten seedlings from each treatment were transplanted to experimental field. Morphological traits, including leaf length, leaf width, plant height, branch length, stem diameter, and number of bunches per plant, were recorded two months later after transplantation.
The fresh leaf material was harvested from the three-month-old plants treated with gamma rays. DNA was extracted using a modified CTAB method [
PCR was carried out in a total volume of 20
Data of seed germination and morphological traits were analyzed using one-way ANOVA with SPSS software package (17.0). Data of ISSR marker analysis were scored as discrete variables, using “1” for presence and “0” for absence of bands for each of the primer pairs. The faint and unclear bands were not considered for data scoring. The binary data generated were used to estimate levels of polymorphism by dividing the polymorphic bands by the total number of scored bands. A dendrogram based on Jaccard’s similarity coefficients was constructed by using Unweighted Pair Group Method with Arithmetic Mean (UPGMA) with the SHAN module of NTSYS-PC 2.0 to show phenetic representation of genetic relationships as revealed by the similarity coefficient.
In this study, we measured the effect of different doses (control, 20, 40, 60, 80, 100, 120, and 140 Kr) of gamma irradiation on seed germination and seedling morphological traits. The results (mean ± standard deviation) are given in Table
Effects of gamma radiation on seed germination percentage and morphological traits of
Dose | SG (%) | LL (cm) | LW (cm) | SH (cm) | BL (cm) | SD (mm) | NB |
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20 Kr |
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40 Kr |
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60 Kr |
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80 Kr |
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100 Kr |
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120 Kr |
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140 Kr |
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Each value was expressed as mean ± standard deviation. SG, seed germination; LL, leaf length; LW, leaf width; SH, seedling height; BL, branch length; SD, stem diameter; NB, number of branches per plant. Within columns, means ± standard deviation followed by the same letter are not significantly different at
Seeds untreated with gamma rays showed higher germination rate than the irradiation treatment except for the 20 Kr dose treatment with germination percentage of 45%. In the treated seeds, the germination decreased significantly (
Only the seedling height showed a constant decline as the irradiation doses increased, though not reaching the significant level. Similar to the effect on germination, the branch length decreased gradually as the irradiation dose increased up to 120 Kr and showed a slight rise at 140 Kr treatment. The maximum stem diameter (3.93 mm) and number of branches per plant (12) occurred at 80 Kr dose treatment, while the minimum stem diameter (2.79 mm) and number of branches per plant (7.33) were observed at 120 Kr dose treatment.
In our research, the differences among the gamma irradiation mutants were examined by using ISSR marker. 20 primers were selected for amplification and data scoring (Table
List of primers, number of amplified products, polymorphic bands, and polymorphism percentage.
Number | Primers | Sequence (5′–3′) | TAP | NPB | PPB (%) |
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1 | UBC857 | AGAGAGAGAGAGAGAGYG | 17 | 12 | 70.59 |
2 | UBC818 | CACACACACACACACAG | 7 | 2 | 28.57 |
3 | UBC850 | GTGTGTGTGTGTGTGTYC | 9 | 4 | 44.44 |
4 | UBC840 | GAGAGAGAGAGAGAGAYT | 10 | 2 | 20.00 |
5 | UBC812 | GAGAGAGAGAGAGAGAA | 7 | 1 | 14.29 |
6 | ISSR04 | GAGAGAGAGAGAGAGACT | 10 | 2 | 20.00 |
7 | ISSR11 | ACACACACACACACACTG | 11 | 8 | 72.73 |
8 | ISSR21 | GAGAGAGAGAGAGAGATG | 7 | 2 | 28.57 |
9 | ISSR25 | ACACACACACACACACCT | 10 | 4 | 40.00 |
10 | ISSR24 | ACACACACACACACACCA | 13 | 7 | 53.85 |
11 | ISSR36 | GCGTCTCTCTCTCTCTC | 8 | 2 | 25.00 |
12 | ISSR47 | AGAAGAAGAAGAAGAAGAAGA | 7 | 4 | 57.14 |
13 | ISSR02 | AGAGAGAGAGAGAGAGCC | 12 | 10 | 83.33 |
14 | ISSR05 | GAGAGAGAGAGAGAGACC | 7 | 5 | 71.43 |
15 | ISSR06 | GAGAGAGAGAGAGAGACG | 12 | 9 | 75.00 |
16 | ISSR08 | GTGTGTGTGTGTGTGTCC | 15 | 14 | 93.33 |
17 | ISSR12 | CCCTCCCTCCCTCCCT | 3 | 1 | 33.33 |
18 | ISSR14 | CTTCACTTCACTTCA | 7 | 3 | 42.86 |
19 | ISSR10 | ACACACACACACACACTA | 4 | 1 | 25.00 |
20 | ISSR15 | GGAGAGGAGAGGAGA | 7 | 1 | 14.29 |
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Total | 183 | 94 | 913.75 | ||
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Mean | 9.15 | 4.7 | 45.69 |
TAP, total amplified products; NPB, number of polymorphic bands; PPB, percentage of polymorphic bands.
A total of 183 bands were scored, of which 94 were polymorphic. The number of bands generated per primer varied from 3 to 17 and a minimum of 3 bands was generated by the primer ISSR12, while the maximum of 17 bands was scored with UBC857 followed by ISSR08 which produced 15 bands (Table
The genetic similarity based on ISSR data ranged from 0.6885 to 1.000, with an overall mean of 0.7884 (Table
Distance matrix based on Jaccard’s coefficients.
Control | 20 Kr | 40 Kr | 60 Kr | 80 Kr | 100 Kr | 120 Kr | 140 Kr | |
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Control | 1.0000 | |||||||
20 Kr | 0.7596 | 1.0000 | ||||||
40 Kr | 0.8579 | 0.7814 | 1.0000 | |||||
60 Kr | 0.7377 | 0.7486 | 0.8033 | 1.0000 | ||||
80 Kr | 0.8361 | 0.7705 | 0.8251 | 0.8251 | 1.0000 | |||
100 Kr | 0.7814 | 0.7596 | 0.8033 | 0.7486 | 0.8142 | 1.0000 | ||
120 Kr | 0.8087 | 0.6885 | 0.8197 | 0.7432 | 0.8306 | 0.7650 | 1.0000 | |
140 Kr | 0.8415 | 0.7541 | 0.8087 | 0.7869 | 0.7978 | 0.7541 | 0.8251 | 1.0000 |
Dendrogram representing the morphological variation among seven irradiated mutants (20, 40, 60, 80, 100, 120, and 140 Kr) and the control based on similarity coefficients of ISSR data.
Many researchers have reported that mutagens, including chemical and physical mutagens, can interact with cellular molecules, particularly water, to produce free radicals (H, OH). These free radicals could combine to form toxic substances, such as hydrogen peroxide (H2O2), which indirectly lead to the destruction of cells [
Many researchers reported that gamma rays treated plants can change the vegetative traits, rhizome characteristics, and flowering development and its maturity in either a positive direction or a negative direction [
That is, this progressive reduction in
Mutation breeding is based on induction of mutation, genetic diversity evaluation, and screening and molecular identification of desired character. During this process, molecular markers have made great contributions in the genetic characterization, screening of mutant plants, and the molecular identification of specific agronomic trait. Among various molecular markers, ISSR is easy to apply, highly informative, reliable, repeatable, and inexpensive [
The authors declare that there are no conflicts of interest regarding the publication of this paper.
This work was funded through projects of the National Natural Science Foundation of China (31260572 and 71263012), the Science and Technology Innovation Talent Team Construction of Guizhou Province (