Analysis Among 13 Species Of Primula.L In South-west Sichuan Of China Using RAPD Marker

The RAPD (randomly amplified polymorphic DNA) marker was applied to studying the genetic diversity and phylogenetic relationship among thirteen Primula species in southwestern areas of Sichuan province, such as Longchi, Kangding, Mugecuo and Litang. P.yargongensis,P.alpicola and the endemic species of Sichuan—P.chienii were evaluated for the first time, and the phylogeny status of P.secundiJlora was analyzed. Meanwhile, effect factors of the genetic diversity in Primula were discussed, and corresponding protection strategies and research directions in the future were suggested.The results indicated that:1 Optimized method and reaction system of RAPD in Primula were established. The optimum reaction system were as follows: 10×Buffer (including Mg²⁺) 2μl, dNTP 150μmol/L, Taq enzyme0.4μl, DNA30～40μg/μl, Primer2.5μmol/L.The optimum PCR reaction method were as follows: 95℃predegenerate 5min, 94℃degenerate 30s, 35℃Cregenerate 50s, 72℃elongate 90s, 40 circles in all, and the last circle maintain 5min at 72℃.2 The RAPD analysis results of 85 materials in 13 Primula showed that: 250 DNA bands were amplified with 28 primers, including 209 polymorphic bands, account for 83.6%, which indicated the genetic diversity of Primula species was relatively high. 173 DNA bands were amplified in P.yargongensis, including 82 polymorphic bands which account for 47.4%; 206 DNA bands were amplified in Palpicola, including 155 polymorphic bands, account for 75.2%; 187 DNA bands were amplified in P.chienii, including 94 polymorphic bands, account for 50.3%.3 The results of genetic diversity analysis in P.yargongensis populations showed that: the genetic distance of 17 materials was between 0.0345～0.9523, and the average genetic distances among materials was 0.4584. Among populations, the average genetic distance and genetic diversity of materials from Mugecuo population was higher, and there existed greater genetic differentiation among materials. However, the genetic distance of materials from Kangding and Litang population was lower, and the genetic differentiation was smaller, so the genetic diversity was lower. Among populations, the average genetic distance of Mugecuo and Kangding population were relatively lower, which indicated that the relationship between these two populations was closer; while the average genetic distance of Kangding and Litang population were relatively higher, which indicated that the differentice and the relationship between these two populations was further. The genetic differentiation among populations enlarged with the expanding of locations.4 The analysis results of P.alpicola population showed that: there were distinct genetic differences among different colors of P.alpicola, and genetic differentiation and genetic difference among different blue P.alpicola were significant. The results also showed that the genetic diversity in P.chienii was low, and this might be the suspected reason for narrowly distribution in P.chienii.5 The effects on genetic diversity of Primula in different environmental and eco-geographic areas were compared. The results showed that: well-developed environmental areas had the advantages to improving the genetic diversity of Primula, and the high level and stable gene flow in the same areas had the advantages to making Primula populations reach the same level. The Primula populations in Longchi, the western of Sichuan basin, were clustered together. However, the Primula populations from Ganzi had a higher genetic diversity, and its genetic difference was more distinct than that from Longchi. Therefore, the genetic difference of Primula was affected by different eco-geographic areas.6 As cluster analysis with RAPD markers was partially similar to morphological type. Primula pulverulenta Duthie, which belongs to Sect. Proliferae from the viewpoint of morphological type, was not clustered together with other two groups of Sect. Proliferae, but clustered together with Primula populations in Longchi, which indicated that the genetic structure of Primula was strongly affected by different eco-geographic areas. By discussing the phylogeny status of P.secundiflora, P.secundiflora was clustered together with Sect. Proliferae, which showed that it had a closer relationship with Sect. Proliferae.