Single sequence repeat（SSR） multiplexing is a semi high-throughput PCR methodology for the analysis of multiple SSRs.We developed two SSR multiplexes selected from SSR loci previously reported in the pine literature...Single sequence repeat（SSR） multiplexing is a semi high-throughput PCR methodology for the analysis of multiple SSRs.We developed two SSR multiplexes selected from SSR loci previously reported in the pine literature and tested the transferability of both SSR multiplexes in nine other pine species.We tested 234 nuclear SSR loci（n SSRs） previously described in the pine literature and selected ten n SSRs following the simple criteria of interpretability and reproducibility.Selected nuclear loci were divided into two n SSRs multiplex sets and their amplification was optimized for three different multiplex PCR methods based on：（a） a custom PCR protocol,（b） a custom protocol with hotstart taq polymerase,and（c） a commercially available kit for SSR multiplexing.To validate their performance,the level of genetic diversity was assessed in three Scots pine natural populations（Hungary,northern Sweden and southern Sweden）.In addition,we also tested the transferability of these multiplexes in nine other pine species.We have developed two n SSRs multiplexes of five loci each that will contribute to reduce the costs of n SSR scoring,while increasing the capacity of n SSR loci analysis.Amplification was successful in all three populations（94 % success） and the level of polymorphism（7.1 alleles/marker） was similar to that previously reported for other Scots pine natural populations.Transferability of both multiplexes was successful for those pine species closely related to Scots pine.展开更多
Potato variety Kexin18 was used as testing materials in this research to study the influence on main components in multiplex PCR system, different primer ratios and annealing temperatures in SSR marker amplification. ...Potato variety Kexin18 was used as testing materials in this research to study the influence on main components in multiplex PCR system, different primer ratios and annealing temperatures in SSR marker amplification. Concentration and gradient experiments for four components （enzyme, MgCl2, DNA template and dNTPs） in PCR system were used in the research with the concentration of the other component remained the same; the orthogonal design L9 （34） was applied in the optimization of four sets of primers （STM0014, Pat, SSI, and UGP） in the reaction system at three levels; the temperature gradient selection was used to find out the optimum annealing temperature for the primer. The optimized multiplex PCR system of potato SSR marker with a total volume of 20 μL ： 2.5 μL 25 mmol.L-1 MgCl2, 0.6 μL 10 mmol·L-1 dNTPs, 0.8 U Taq, 80 ng DNA template was ultimately established through the comparison and analysis of test results; the ratio of four pairs of 4 mmol. L1 primers was 2 ： 1 ： 2 ： 3, and the annealing temperature was 54.7℃. The optimized reaction system could be repeated stably; and the stable and reliable amplification results were able to clearly distinguish different potato varieties. This research built the solid foundation for the further study of genetic diversity of potato germplasms and construction of DNA fingerprinting..展开更多
为了建立稳定可靠的玉米真实性和纯度鉴定SSR标记,对DNA提取方法、SSR引物和多重PCR反应程序进行了优化。结果表明,用预热到75℃以上的研钵和95℃的1.5×CTAB提取缓冲液进行材料研磨,可得到纯度高、完整性好的DNA,并且提取成本较低...为了建立稳定可靠的玉米真实性和纯度鉴定SSR标记,对DNA提取方法、SSR引物和多重PCR反应程序进行了优化。结果表明,用预热到75℃以上的研钵和95℃的1.5×CTAB提取缓冲液进行材料研磨,可得到纯度高、完整性好的DNA,并且提取成本较低。利用软件Primer Premier 5.0和Oligo 6.72对玉米指纹鉴定的SSR核心引物进行重新分析与设计,建立了21对SSR通用引物构成的8组多重PCR复合扩增体系和3步法扩增程序,均能在统一的PCR扩增条件下进行,扩增片段之间不存在交叉现象,扩增条带清晰,扩增结果稳定,这一扩增体系检测效率比单对SSR引物提高2.6倍以上。展开更多
基金supported by GENECAR and Kempe foundation through the Research School in Forest GeneticsBreeding at the Swedish University of Agricultural Sciences(SLU),Umea,Sweden
文摘Single sequence repeat（SSR） multiplexing is a semi high-throughput PCR methodology for the analysis of multiple SSRs.We developed two SSR multiplexes selected from SSR loci previously reported in the pine literature and tested the transferability of both SSR multiplexes in nine other pine species.We tested 234 nuclear SSR loci（n SSRs） previously described in the pine literature and selected ten n SSRs following the simple criteria of interpretability and reproducibility.Selected nuclear loci were divided into two n SSRs multiplex sets and their amplification was optimized for three different multiplex PCR methods based on：（a） a custom PCR protocol,（b） a custom protocol with hotstart taq polymerase,and（c） a commercially available kit for SSR multiplexing.To validate their performance,the level of genetic diversity was assessed in three Scots pine natural populations（Hungary,northern Sweden and southern Sweden）.In addition,we also tested the transferability of these multiplexes in nine other pine species.We have developed two n SSRs multiplexes of five loci each that will contribute to reduce the costs of n SSR scoring,while increasing the capacity of n SSR loci analysis.Amplification was successful in all three populations（94 % success） and the level of polymorphism（7.1 alleles/marker） was similar to that previously reported for other Scots pine natural populations.Transferability of both multiplexes was successful for those pine species closely related to Scots pine.
基金Supported by the International Cooperation Project of Heilongjiang Science and Technology Department (WC05B08)
文摘Potato variety Kexin18 was used as testing materials in this research to study the influence on main components in multiplex PCR system, different primer ratios and annealing temperatures in SSR marker amplification. Concentration and gradient experiments for four components （enzyme, MgCl2, DNA template and dNTPs） in PCR system were used in the research with the concentration of the other component remained the same; the orthogonal design L9 （34） was applied in the optimization of four sets of primers （STM0014, Pat, SSI, and UGP） in the reaction system at three levels; the temperature gradient selection was used to find out the optimum annealing temperature for the primer. The optimized multiplex PCR system of potato SSR marker with a total volume of 20 μL ： 2.5 μL 25 mmol.L-1 MgCl2, 0.6 μL 10 mmol·L-1 dNTPs, 0.8 U Taq, 80 ng DNA template was ultimately established through the comparison and analysis of test results; the ratio of four pairs of 4 mmol. L1 primers was 2 ： 1 ： 2 ： 3, and the annealing temperature was 54.7℃. The optimized reaction system could be repeated stably; and the stable and reliable amplification results were able to clearly distinguish different potato varieties. This research built the solid foundation for the further study of genetic diversity of potato germplasms and construction of DNA fingerprinting..
文摘为了建立稳定可靠的玉米真实性和纯度鉴定SSR标记,对DNA提取方法、SSR引物和多重PCR反应程序进行了优化。结果表明,用预热到75℃以上的研钵和95℃的1.5×CTAB提取缓冲液进行材料研磨,可得到纯度高、完整性好的DNA,并且提取成本较低。利用软件Primer Premier 5.0和Oligo 6.72对玉米指纹鉴定的SSR核心引物进行重新分析与设计,建立了21对SSR通用引物构成的8组多重PCR复合扩增体系和3步法扩增程序,均能在统一的PCR扩增条件下进行,扩增片段之间不存在交叉现象,扩增条带清晰,扩增结果稳定,这一扩增体系检测效率比单对SSR引物提高2.6倍以上。
