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Objective In this paper, the genetic diversity of 64 samples of Tetrastigma hemsleyanum germplasm resources in Chinese was analyzed. Methods ISSR-PCR was firstly used to amplify, and then POPGENE 32 software and NTSYS software was used to analyze the genetic diversity and phylogenetic relationship of 64 samples of T. hemsleyanum germplasm resources, and phylogenetic tree was constructed according to the UPGMA method. Results Ten primers with clear and reproducible bands were screened from 30 primers and used for genomic DNA amplification of 64 sample materials. A total of 83 polymorphic locis were amplified, whose polymorphic percentages were 71.43%-100% and average polymorphism percentage was 94.31%. The amplification polymorphic locis of primer S17 were the most (11) and the amplification polymorphic locis of primer P6 were the least (5), the average amplified polymorphic locis of 10 primers were 8.3. Genetic diversity analysis showed that the average number of alleles (Na) of 64 samples was 1.943 1, the average effective allele number (Ne) was 1.381 08, the average Nei’s gene diversity index (H) was 0.242 98, and the average Shannon diversity index (I) was 0.385 83. The variation range of the genetic similarity coefficient of the 64 samples was 0.431 8-0.988 6. A total of 64 samples were divided to six groups by UPGMA clustering method according to the similarity coefficient matrix when the genetic similarity coefficient was 0.715 5, which showed the abundant genetic diversity and relative gene stability of T. hemsleyanum germplasm resources. In addition, amplification figures by primers ISSR20, UBC857, and S17 were screened based on amplification result of 10 ISSR primers, and DNA fingerprinting was constructed, which can be used to identify 64 samples of T. hemsleyanum tested. Conclusion There are abundant genetic diversity and relative gene stability in T. hemsleyanum germplasm esources in China. ISSR analysis can reveal the genetic relationship among T. hemsleyanum germplasm resources in China, and provide certain reference for the evaluation, identification and new variety breeding of T. hemsleyanum germplasm resources in China.
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Objective To understand the genetic diversity and genetic relationship of 64 samples of Tetrastigma hemsleyanum germplasm resources. Methods Fluorescently labeled SSR were used for PCR amplification, POPGENE32 software was used to analyze genetic diversity and genetic relationship of 64 samples of T. hemsleyanum germplasm resources, UPGMA method was used to construct their genetic tree map, NTSYS software was used to construct their two-dimensional principal component analysis map and three-dimensional scatter map. Result Eight pairs of primers with clear and reproducible bands were screened from 14 pairs of primers and used for genomic DNA amplification of 64 sample materials. The observed number of alleles (Na) ranged from 3 to 13, and the mean value was 7.875 0; The effective number of alleles (Ne) ranged from 1.424 9 to 6.087 4, and the mean value was 3.605 2; The Shannon information index (I) ranged from 0.689 5 to 2.082 4, and the mean value was 1.424 0; The observed heterozyghosity (Ho) ranged from 0.206 3 to 0.734 4, and the mean value was 0.524 7; The expected heterozygosity (He) ranged from 0.300 6 to 0.842 4, and the mean value was 0.658 4; The Nei’s gene diversity (H) ranged from 0.298 2 to 0.835 7, and the mean value was 0.653 2; The polymorphism information content (PIC) ranged from 0.288 0 to 0.817 5, and the mean value was 0.614 5; The genetic similarity coefficient ranged from 0.115 4 to 0.954 5; The genetic distance ranged from 0.000 0 to 3.218 1. It was indicated that the genetic relationship of 64 T. hemsleyanum germplasm resources was far, and the degree of genetic differentiation was high. At the genetic distance of 1.018 9, 64 T. hemsleyanum germplasm resources could be divided into five groups. Conclusion There was no necessary connection between geographical difference and genetic difference of germplasm. Themsleyanum germplasm resources are rich in genetic diversity. The results of fluorescently labeled SSR analysis can provide some references for the utilization and variety breeding of T. hemsleyanum germplasm resources.
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Objective: The physiological regulation mechanism of seedlings germinated from Jiangxi local yam (Ruichang yam, Nancheng yam, Yongfeng yam, and Guangfeng yam) microtubers under salt stress was studied and the salt tolerance of seedlings germinated from microtubers of four kinds of local yams in Jiangxi was compared, which provided some references for salt tolerance mechanism research and salt tolerance breeding of four local yams in Jiangxi. Methods: Some physiological and biochemical index detection of seedlings germinated from Jiangxi local yam microtubers under salt stress were studied by spectrophotometer method. The subordinate function, principal component analysis (PCA), and clustering analysis of their salt tolerance were accomplished by fuzzy mathematics subordinate function formula and SPSS 19.0 software, respectively. Results: The total chlorophyll content and root activity of seedlings germinated from Jiangxi local yam microtubers under 0-300 mmol/L salt stress decreased significantly. The soluble protein content of seedlings germinated from Ruichang yam, Nancheng yam, and Guangfeng yam microtubers under 0-300 mmol/L salt stress increased firstly and then decreased, while the soluble protein content of seedlings germinated from Yongfeng yam microtubers under 0-300 mmol/L salt stress decreased significantly. The total soluble sugar content, proline content, MDA content and membrane permeability of seedlings germinated from Jiangxi local yam microtubers under 0-300 mmol/L salt stress significantly increased. The POD, SOD, and CAT activities of seedlings germinated from Jiangxi local yam microtubers under 0-300 mmol/L salt stress firstly increased and then decreased. Based on the subordinate function and PCA of SPSS 19.0 software, three principal components of 10 physiological and biochemical indexes of seedlings germinated from Jiangxi local yam microtubers under salt stress were induced. The salt tolerance order of seedlings germinated from Jiangxi local yam microtubers was Guangfeng yam > Yongfeng yam > Ruichang yam > Nancheng yam. Through the cluster analysis by SPSS 19.0 software, the salt resistance of four kinds of Jiangxi local yam is divided into two types, the salt tolerance of Guangfeng yam and Yongfeng yam was strong, and of Ruichang yam and Nancheng yam were sensitive to salt stress. Conclusion: The regulation mechanism of salt tolerance of seedlings germinated from four kinds of Jiangxi local yam microtubers is revealed and the objective evaluation of salt tolerance of four kinds of Jiangxi local yam is made in the paper, which will provide a theoretical basis for sowing in the field of four kinds of Jiangxi local yam microtubers.
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Objective: To analyze the genetic diversity of 11 varieties of Dioscorea opposita germplasm resources from Jiangxi, and detect the genetic stability of the tissue cultured plantlets. Methods: RAPD-PCR was applied. Results: Twenty primers were screened out, 238 bands were amplified, 234 of them were polymorphic bands, the average amplified polymorphic bands were 11.7, and the ratio of polymorphic bands was 98.32%. The genetic similarity coefficient was 0.575 6-0.970 6 and the average similarity coefficient was 0.723 1. According to the results of UPGMA clustering analysis, the 11 D. opposita germplasm resources from Jiangxi province were divided into five categories: The first class included D. bulbifera. The second class included D. opposita. The third class included D. opposita Nancheng 1, D. opposita, D. opposita, D. opposite, and D. opposita, which showed that D. opposita 1, D. opposita, D. opposita and D. opposite, had closer relationship with D. opposita came from Wenxian County, Jiaozuo City, Henan province. The fourth class included D. opposita, D. opposita Nancheng 2 and D. opposita. The fifth class included D. opposita. The RAPD analysis results of plantlets subcultured for six times by in vitro rapid propagation method of stem segment with a bud of D. opposita L. germplasm resources from Jiangxi province (treatment group) and their seedlings germinated from microtuber (control group) also showed that 11 yam varieties were spread out 126-179 identifiable bands and each variety appeared 5-18 mutation bands, which indicated plantlets subcultured for six times by in vitro rapid propagation method of stem segment with a bud of D. opposita. germplasm resources from Jiangxi province existed somaclonal variation, but their genetic similarity coefficient compared with their seedlings germinated from microtuber was still relatively high, illustrating that their genetic traits were still relatively stable and did not affect their genotype. Conclusion: The experimental results can provide the reliable basis for introduction breeding, resource improvement, variety identification, germplasm conservation, and their plantlet culture of D. opposita germplasm resources from Jiangxi province.
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Objective: In order to provide a theoretical basis for clarifying the mechanism of synthesis of saponins during different induction formation period of Dioscorea bulbifera microtubers, squalene synthase (SQS) gene expression of D. bulbifera microtubers was analyzed in this paper. Methods: Using Actin gene as a reference gene, Real-time quantitative PCR (qRT-PCR) analysis technology was applied. The amplification curve and solubility curve of SQS (target gene) and Actin (reference gene) were successfully constructed by SYBR Green I after RNA was extracted from different formation period of D. bulbifera microtuber and cDNA was obtained by reverse transcription. Result: Quantitative results showed that: During the initial stage (18-36 d) of D. bulbifera microtuber formation, the expression level of SQS gene increased significantly. During the mid-term (36-72 d) of D. bulbifera microtuber formation, the expression level of SQS gene decreased significantly, and tended to be stable. During the later stage (72-90 d) of D. bulbifera microtuber formation, the expression level of SQS gene decreased significantly again. Conclusion: In general, in the process of D. bulbifera microtuber formation, the expression level of SQS gene shows the trend of "low-high-low-constant-low", which indicates that SQS is a key enzyme of saponin biosynthesis during the different induction formation period of D. bulbifera microtubers.
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Objective: To discuss the influence of several factors on the cryopreservation of embryogenic calli induced from Dioscorea bulbifera microtuber by droplet-vitrification and to test the genetic stability of the regenerated plantlets after freezing from the aspects of morphology, physiology, DNA content, as well as the photosynthetic characteristics and chlorophyll fluorescence parameters in this paper. Methods: Plant tissue culture (including microtuber induction and embryogenic callus induction), plant physiology index detection (including total chlorophyll, soluble protein, soluble sugar and superoxide dismutase enzyme and peroxide enzyme activity), and cell flow cytometry were applied. Results: The best cryopreservation conditions of embryogenic callus of D. bulbifera microtuber were as following: Embryogenic calli were precultured in liquid media of MS+KT 2 mg/L+NAA 0.5 mg/L+2, 4-D 0.5 mg/L+0.3 mol/L sucrose for 1 d and then treated in loading liquid (MS+2 mol/L glycerol+0.4 mol/L sucrose, pH 5.8) for 20 min. In order to dehydrate, embryogenic calli were transferred in 100% PVS2 at 0℃ for 40 min. After dehydration, the embryogenic calli were inoculated to PVS2 small drops in the aluminum foil strips and then dipped in liquid nitrogen (LN). Finally the aluminum foil strips were quickly transferred to freezing tube that filled with LN and then put into LN tank. After conserving for 1 d in LN, the aluminum foil strips were removed and the embryogenic calli were immersed into liquid washing media (MS+KT 2 mg/L+NAA 0.5 mg/L+2, 4-D 0.5 mg/L+1.2 mol/L sucrose, pH 5.8) preheated in 37℃ warm water. After separated from the aluminum foil strips, the embryogenic calli were washed with fresh liquid washing media at room temperature for there times, 10 min each time. After washing, the embryogenic calli were transferred onto differentiation medium (MS+KT 2 mg/L+NAA 0.5 mg/L+30 g/L sucrose+5 g/L agar), and cultured in dark for 2 d and then cultured in 12 h/d photoperiod, the cell survival rate reaches above 89%. The morphological and physiological indexes and the content of DNA of two kinds of plantlets, which regenerated from cryopreserved and non-cryopreserved embryogenic calli induced from D. bulbifera microtuber by droplet-vitrification, showed no significant difference (P>0.05). Conclusion: Cryopreservation technology system of embryogenic calli induced from D. bulbifera microtuber by droplet-vitrification is established and the regeneration plants have no genetic variation, which provides the theoretical basis and technical basis for the long-term preservation of germplasm resources in the plants of Dioscorea L.