| Elymus sibiricus and Medicago falcata L. are excellent herbages which can resist drought, salt, cold and have higher crude protein, and play an important role in genetical breeding as rich wild resources. Based on the previous studies conducted by our laboratory, the genetic diversity of both Elymus sibiricus and Medicago falcata L. was analyzed by simple sequence repeat(SSR) markers, esterase isozyme markers and amplified fragment length polymorphism(AFLP) markers, and methylation level was done under stress using methylation-sensitive amplified polymorphism(MSAP) techniques. The results were as follows:1. SSR marker analysis of 20 drought-stressed Elymus sibiricus: the number of total bands amplified by 10 primer pairs was 115, and the bands ranged from 500 to 2000 bp in size, among which there were 103 polymorphic bands, their percentage was 89.6%, the average polymorphism information content(PIC) was 0.368, SSR marking index was 3.87, the genetic similarity coefficient of them was from 0.39 to 0.85, their genetic distance was from 0.16 to 0.94. These data all showed that Elymus sibiricus has rich genetic diversity. UPGMA clustering showed that the accessions from the same region tended to be divided into one branch but it had special case. The clustering result was quite different between drought-stressed materials and untreated ones.2. Esterase isozyme analysis of 20 drought-sressed Elymus sibiricus: 20 Elymus sibiricus materials exhibited 17 zymograms by denaturing polyacrylamide gell electrophoresis(PAGE), and most of the zymograms were different. Among the 17 zymograms there were 16 polymorphic zymograms, its percentage was 94.1%, its mobility was 0.154 to 0.692, 0.462-mobility band was a common band for 20 Elymus sibiricus materials, this demanstrated that these materials were homologous, so the common band could be a fundamental band for the tested materials. On the common band, different material had different degree in dyeing which displayed that their enzyme activity and content was different. This reflected polymorphism between the materials. The genetic similarity coefficient among them was from 0.01 to 0.93, so they had rich genetic polymorphism, the clustering result had much to do with their origin, the accessions from the same region tended to be divided into one branch but it had special case.3. MSAP assay of drought-sressed Elymus sibiricus: 2 Elymus sibiricus materials which had better polymorphism were choosed to perform MSAP assay. Based on the results of SSR analysis and esterase isozyme analysis, 475 fragments were amplified by 15 pairs of selective primers, of which 376 fragments were methylated, its percentage in total fragments was 79; 2 accessions of Elymus sibiricus were treated by drought stress at various intervals, 0 day, 4 days, 6 days and 8 days, the average proportion of polymorphism(P) were 57.6%, 64.1%, 69% and 77.6%, respectively; compared with the control, the total methylation polymorphism of Elymus sibiricus treated by drought stress for 4 days, 6 days and 8 days was 8%, 14.4% and 23%, respectively, indicating the levels of DNA methylation and methylation polymorphism increased gradually accompanied with the enhancement of drought stress.4. AFLP marker analysis from 20 populations of Medicago falcata L.: 533 fragments were amplified by 15 pairs of selective primers, of which 396 bands were polymorphic, its percentage was 74.3%, the average polymorphism information content was 0.364, AFLP marking index was 9.589, Nei’s genetic diversity was 0.268, Shannon’s information index was 0.399, the genetic similarity coefficient of them was from 0.65 to 0.81, their genetic distance was 0.208 to 0.430, which pointed out that there was a certain difference in the AFLP pattern of the tested materials and they had rich genetic polymorphism, UPGMA clustering proved that the accessions from the same region tended to be divided into one branch but it had special case.5. AFLP marker analysis of individual Medicago falcata L. materials: 18 Medicago falcata L. resources were amplified by 15 pairs of selective primers, 493 bands were founded, of which 376 bands were polymorphic, its percentage was 76.3%, average polymorphism information content was 0.328, AFLP marking index was 8.19, Nei’s genetic diversity was 0.246, Shannon’s information index was 0.372, the genetic similarity coefficient of them was from 0.655 to 0.873, their genetic distance was 0.136 to 0.423. Therefore, it could be found that the data of AFLP analysis of individual Medicago falcata L. materials were lower than that of population materials, which indicated that the individual materials in one population had consistent physiological and ecological conditions and they had small genetic polymorphism, UPGMA clustering revealed that the accessions from the same region tended to be divided into one branch but it had special case.6. MSAP analysis of 12 Medicago falcata L. materials: 10 primer pairs which have higher polymorphism and better repeatability were selected from 25 primer pairs to amplify tested materials, the result was that 190 polymorphic bands were found, of which 130.75 fragments were methylated, its percentage in total fragments was 55.6%, the percentage of full methylated bands in methylated bands was 62.9%, that disclosed there was a certain difference in the methylation pattern of the tested materials and the figure was higher than other plants’ genome, which showed species divergence in epigenetic.7. MSAP analysis of low tempreture-stressed Medicago falcata L. materials: 10 primer pairs which have higher polymorphism and better repeatability were selected from 25 primer pairs to amplify tested materials, the result was that 189 polymorphic bands were found, of which 154 fragments were methylated, its percentage in total fragments was 81.6%, the percentage of full methylated bands in methylated bands was 76.5%. The level of DNA methylation increased obviously accompanied with low temperature-stress. The result explained that the DNA methylation took part in the regulation of gene expression of low temperature-stressed Medicago falcata L.
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