Construction Of DNA Fingerprintings In Rapeseed Varieties (B. Napus L.) Using Multiplexed Fluorescent SSR Markers

The construction of rapeseed DNA fingerprinting is of important application value in rapeseed purity and identity testing, variety right' protection, variety quality inspection in regional trial, and is of great significance in heterotic group germplasm innovation and variety breeding. The research based on capillary electrophoresis with fluorescent SSR markers, a high throughput detection system fitting for rapeseed variety identification was established finally. The system would be applied in rapeseed variety identification and establishing DNA fingerprinting of great scale rapeseed variety. This study is to establish the technical platform of rapeseed standard DNA fingerprint through the key technic of experimental screening of primer pairs, basic process of establishing common multiplex PCR combination and fluorescent multiplex PCR combination.The main results were summarized as follows:(1) According to high-density Brassica linkage maps and previous screening results for the primer in our laboratory,52 pairs of SSR primers with high polymorphism and amplification quality were selected initially. The 5'end of left and right primers were both jointed with M13F and M13R, respectively, and 24 of them can be selected for multiple PCR combination by polyacrylamide gel electrophoresis. At the same time record the relative size of the products.(2) I carried out research about the basic process of establishing multiplex PCR using selected multiplex PCR primer combinations. The upstream universal primer of M13F was labeled with 6-FAM, HEX, respectively. A fully-formed multiplex system fitting for PCR amplification reaction and detection was established finally through the regulation of PCR composition and reaction conditions. The amplified products were detected using ABI 3500 Genetic Analyzer. Furthermore, the genotype of each sample was defined according to the length of the amplification products analyzed by GeneMapper Analysis Software 4.1. (3) In this experiment,179 rape cultivars (lines) were genotyped by using screened 16 pairs of SSR primers.2-9 alleles were detected in 16 pairs of SSR primers, with in total of 78 alleles, an average 4.88 alleles per locus. A mean PIC of 0.82, with a range of 0.35-0.98 was observed. Cluster analysis allowed differentiation among the 179 rape cultivars (lines), which, surprisingly, clustering analysis showed that the genetic diversity of rapeseed varieties was relatively narrow.(4) According to the results of multiplex PCR combination, with PIC values and DP, a group of core primer combinations was selected. Finally the DNA fingerprints of 179 rapeseed varieties(B. napus L.) were established by means of 9 pairs of SSR primers. Using primer-in-group method can expand the core primers quickly to identify the purity and reality of Brassica hybrids.