Microsatellite Primers Development By Magnetic Beads Enrichment In Siraitia Grosvenorii

Luohanguo (Siraitia grosvenorii (Swingle) C.Jeffrey), a dioecious species of Cucurbitaceae, is economic important cultivated plant, endemically distributing in Guangxi, China. After years of breeding and improvement, degradation and hybrid between varieties are appearing. SSR, with the virtues of hyper-variability, codominance, and high reproducibility, was a more effective molecular marker in aspects of genetic research.The objectices of this study were to develop microsatellite primers of Siraitia grosvenorii.In this study, the microsatellite primers were obtained by using the method of enrichment by magnetic beads. First of all, the genomic DNA of Siraitia grosvenorii was digested with a restriction enzyme Rsa I, and then ligated SuperSNX24 linkers to the digested fragment. These genomic DNA fragments were hybridized with five kinds of singlestrand biotinylated microsatellite probes (AC) 15, (AT) 15, (CT) 12, (ATC) 10, (CCA) 10. Then the hybrid mixtures were adsorbed by magnetic beads, coated by streptavidin. And the fragments of containing microsatellite DNA wre enriched. Then the fragments enriched were cloned using pMD19-T vector and transformed into competent E. coli DH5a. Screened the positive clones by PCR amplification using M13 universal primers. And primers were designed for microsatellite sequences using program Primer Premier 5.0. Selected the desired primers in denaturing polyacrylamide gel (6%).Finally,11 pairs of microsatellite primers were selected.The 15 pairs of primers were tested for polymorphisms in 98 Siraitia grosvenorii individuals (74 Cultivars and 24 wild individuals). A total of 111 alleles were detected, with an average of 7.4 alleles per microsatellite locus. There were fewer alleles in Sg2 and Sg11,2,3, respectively. There were 12 alleles in Sg4, which was the largest allele number in 15 microsatellite locis. The mean polymorphic information content (PIC) was 0.716 in 15 microsatellite locis. The PIC value of Sg2 was 0.543, the minimum in 15 locis; Sg21 was 0.861, the highest value.In 15 microsatellite locis, recorded respectively 38,31,24,48,46,50 alleles in Qingpiguo, Hongmaoguo, Baopengzi, Dongguahan, Chashanguo and the male, the mean PIC value were 0.266,0.197,0.201,0.419,0.396,0.411, respectively. This demonstrated a lower genetic diversity level in Qingpiguo, Hongmaoguo, Baopengzi, while Chashanguo and Dongguahan showed a relatively high genetic diversity, and the male individuals also had a high level of genetic diversity. Dendrogram was constructed using an unweighted pair-group method with arithmetical averages (UPGMA) method and Nei & Li genetic distance. The results also demonstrated genetic differentiation between cultivated individuals, and indicated the problems of single, mix between varieties were existed. The highest genetic diversity level, which was much higher than the cultivars, was in Jinxiu population.The number of alleles and the value of PIC in 15 SSR locis were 98 and 0.611. By Hardy-Weinbery test, Sg4 and Sg17 deviated from equilibrium (P< 0.05).The results of this study indicated the 15 pairs of microsatellite primers, developed by magnetic beads, got expected amplification effect in Siraitia grosvenorii genomic DNA. And this illustrated the 15 pairs of microsatellite primers could be further used in Siraitia grosvenorii genetic research.