| As the fifth largest food crop in China, sweet potato is also an important raw material for multiple industrial uses, thus of great significance to the national economy. Sweet potato is asexually propagated crop in most parts of China, but can produce flowers and seeds in Hainan area as a result of the unique tropical monsoon. However, the main varieties of sweet potato in Hainan are a little less,and quality indicators of some varieties are relatively low. The development of sweet potato industry which did not show full play to geographical advantages in Hainan (compared with northern region). Therefore, select and breed adaptive sweet potato varieties would greatly promote the related industries in Hainan area. It would be of great importance to collect more sweet potato germplasm resources and clarify their genetic relationships first.In this study, we optimized the SSR-PCR reaction system of sweet potato, a-nd studied the genetic diversity of 30 sweet potato germplasm resources by the optimized SSR molecular marker technology. This study provided a theoretical basis for future selection and breeding of sweet potatos in Hainan.The main results are as follows:(1 ) The single and multiple SSR-PCR reaction systems were established for sweet potato. The reaction system included: 10?L 2×PCR Mix,100ng template DNA, 0.4?mol/L primers, 1?L glycerol, with a total volume of 20?L. The PCR amplification started with initial denaturation for 4 min at 94 ?, followed by 20 cycles of denaturation for 45s at 94 ?, anneal for 30s(annealing time varied depending on the DNA fragment length) at Tm+5?Tm-5 ? (annealing temperature decreased 0.5 ? per cycle, the lower Tm of the primer pair was chosed), extension for 1 min at 72?, then 15 cycles of denaturation for 45s at 94?, anneal for 30 s at Tm-5?, extension for 1 min at 72?. The amplification was completed after extension for 7 min at 72 ?, and then stored at 4 ?. The best sample amount of polyacrylamide gel electrophoresis was 1.5-2p,L. On the basis of single SSR-PCR, the diplex and triplex SSR-PCR system of sweet potato were developed with increased amount of corresponding primers and reduced amount of corresponding ddH2O.For the absent or overloaded bands, concentrations of the corresponding primer can be increased or decreased. The content of Mg2+, dNTP and Taq enzyme could also be properly adjusted according to the primer information.(2 ) By optimized SSR system, the genetic diversity and clustering of 30 sweet potato varieties were analyzied. A total of 118 bands were amplified from 30 primer pairs, with 99 polymorphic bands which accounted 83.9% of the total. One to nine allele bands were amplified by different SSR primer pairs and an average of 3.93 bands were amplified for each primer pair. With the genetic similarity coefficient threshold set as 0.61, the 30 varaties can be divided into two categories. The category I contained 22 varaties of sweet potato and the category ? included 8 varaties. Germplasms with closer relationship clustered more concentrated. The cluster results also accorded with the tuber color but not with the germplasm source areas.(3 ) 13 pairs of SSR primers suitable for multiplex PCR were screened. Among them, the primer pair of C55 was identified as core primer because of its outstanding performance.The molecular fingerprints of 30 sweet potato varieties were successfully constructed by using 6 primer pairs whose resulted clear and easily distinguished bands. As more sweet potato germplasm resources were to be collected,screening of effective SSR primers,especially core primers, needed to be enhanced. |
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