The Analysis Of The Biological Traits And ISSR In Sainfoin Mutants From Seeds Carried Back By "Shijian No. 8" Spaceship

The biological traits and the genetic diversity of Sainfoin dealt with space irradiation were analysed by the survey of the biological traits and ISSR markers. The results were as follows:1. The biological traits of Sainfoin mutants from seeds carried back by"Shijian No. 8"spaceship were studied in 8 biological traits, as height, number of leaflets per compound leaf, leaf size, leaf thickness, leaf color, branch number, plant type and the special variation. The results show that the number of plants of plant type variation was great. 5 biological traits of Sainfoin mutants from seeds carried back by"Shijian No.8"spaceship were studied using SPSS.The results show that significant difference existed in the height, blade thickness and leaf color of Sainfoin mutants treated by space irradiation. However, the variation of Sainfoin mutants in anoter biological traits was not clearly very much, such as the variation of the number of leaflets per compound leaf and blade width. In all of Sainfoin mutants, several variations were discovered on some ones.2. Establishment of ISSR-PCR reaction system for Onobrychis viciaefolia Scop.: An orthogonal design was used to optimize a ISSR-PCR system with 4 factors (dNTP, Taq polymerase, primer and Mg²⁺) at 3 levels plus the concentration of template DNA. The result shows that the optimized ISSR-PCR system for sainfoin was: 2.5μL 10×PCR buffer, 50 ng template DNA, dNTP 0.2 mmol/L, Taq DNA polymerase 1.5 U, primer 0.4μmol/L, Mg²⁺2.0 mmol/L in a total of 25μL reaction solution. The reaction process went as following: the temperature profile used for PCR was 94℃for 3 minutes, followed by 35 cycles of 94℃for 30 seconds, 46℃-60℃for 30 seconds, 72℃for 1minute, and was terminated with a 10 minutes DNA extension step at 72℃,then keep in 4℃.3. The selecting of ISSR primers and amplification of PCR: There were 12 ISSR primers by sieving which can produced DNA polymorphism in 53 ISSR primers. The result shows that the size of all the bands was in 300-1000 bp. In all experimental materials, 228 DNA bands were amplified by 12 ISSR primers, in which 169 bands were polymorphic, polymorphic ratio (PPB) was 74.1%, and the average of bands were 19 by every primer.4. The genetic variation of group was studied by POPGENE32. The percentage of polymorphic loci(P),Nei's gene diversity(h) and Shannon's index(I) in Sainfoin of the control was lower than that of the plants carried by spaceflight. It shows variation of hereditary substance DNA of Sainfoin can be caused by space environment. These results indicated that the space radiation would be an effective method to develop new Sainfoin germplasm.5. The ISSR-based genetic similarity values among all experimental materials accessions ranged from 0.243-0.838. Abundant genetic diversity among Onobrychis viciaefolia Scop. accessions could be revealed by ISSR markers. Based on the UPGMA cluster analysis and PCA, the 29 materials with different biological traits variation may be divided into six groups, most of genetic variation was within the same group. There was a certain relationship between genetic distance and geographic distance among different Sainfoin variants in the experiment.