Development Of SSR Markers And Their Use To Assess Genetic Diversity In Caragana Microphylla Lam.

Caragana microphylla Lam. is perennial forage shrub which is distributed mostly in Northeastern China, Inner Mongolia, Hebei, Shanxi, Shaanxi, Shandong etc. also in Mongolia and Siberia. Because it's drought resistance; cold resistance and resistant to soil impoverishment, it becomes favorable forage shrub and ecological protection pioneer plant in Northeastern and Northwestern China. Thus it has great potential for development and utilization.Ten populations of Caragana microphylla Lam., which came from central and eastern Inner Mongolia, were used in the experiment. The materials were provided by Institute of Animal Sciences, Chinese Academy of Agricultural Sciences. The work mainly included:1) to develop the polymorphic SSR primers for Caragana microphylla Lam. using ISSR-PCR technology.2) to establish and optimize SSR-PCR system and Filtrate the SSR primers using the optimized SSR-PCR system. 3) to analyze the genetic diversity using SSR markers. The results were as followed.1. Fouty three pairs of SSR primers were designed based on the four ISSR primers, S1:(AG)10, S2:(AC)10, S3:(GA)10, S4:(CA)10. And the average success rate was 11.17%. Our results also indicated that there were large numbers of (AG) n fragments and less of (AC) n fragments in the genome of Caragana microphylla Lam. Compared to the traditional method of SSR marker development, this technique was more efficient. It must be noted that there are twice sequencing and three times primer synthesis.2. Orthogonal design was applied to optimize the SSR-PCR system on Caragana microphylla Lam., which employed four levels and five factors (DNA, Mg, dNTP, primers, and Taq DNA polymerase, respectively). The optimum SSR reaction included 40 ng template DNA,3 mmol/LMg,300μmol/L dNTP,0.6μmol/L primer, 1 U Taq DNA polymerase, 1×Buffer with total 25μL reaction volumes. The PCR procedure was an initial step of 94℃for 10min, followed by 10 cycles of 94℃for 45s,65℃for 1min (decreasing 1℃every cycle),72℃for 1min; 30 cycles of 94℃for 45s,55℃for 1min,72℃for 1min, and a final extension at 72℃for 10 min,4℃hold.3. Choosing the developed SSR primers by the optimized system,37 pairs (86.05%) had target fragments,21pairs (56.76%) had diversity. The rest, there are 5 primers gained non-target fragments and only 1 primer didn't gain fragment. When designing the primers, the gene mutation fragments were chosen may cause the result.4. Ten out 21 polymorphic primers were selected for genetic diversity research. GelQuant, GenAlEx6 and EXCEL were used to analyze the Electrophoresis profile of SSR-PCR. There were 175 alleles detected, the expected heterozygosity (He) were between 0.602 and 0.859; Higher genetic diversity was both observed at loci and populations (He were between 0.683 and 0.830, mean was 0.771); Based on the mean Fst values between 10 populations (9.5%), most of genetic variation of Caragana microphylla Lam. was within population 90.5%, and the gene among population Nm=2.391; Based on UPGMA dendrogram of Caragana microphylla populations, most of populations except the Horqin and Ewenkezuzizhi were gathered by geographic distance.