摘要
【目的】建立稳定性好、可重复性强、多态性扩增效果好的‘库尔勒香梨’SCoT-PCR反应体系,证明SCoT分子标记可以用于梨属植物优良营养系的鉴定。【方法】以新疆库尔勒农二师29团、33团和34团的‘库尔勒香梨’优良营养系为试材,通过L25(56)正交试验设计对‘库尔勒香梨’SCoT-PCR反应体系进行筛选,并对扩增反应程序进行优化,运用正交设计直观分析法和正交设计助手Ⅱ软件对正交设计扩增结果进行分析,从筛选出的20个引物中随机选择2条引物对24份材料扩增,用DNAMAN、Editseq、ORF Finder和NCBI-BLAST-tblastx对测序结果分析。【结果】优化后的香梨SCoT-PCR 20μL反应分析体系含(2.5 mmol·L^(-1)10×buffer with Mg^(2+))2.5μL、Taq酶1.5 U、引物10μmol·L^(-1)、模板DNA25 ng、dNTPs 1.6 mmol·L^(-1)。反应程序为94℃预变性4 min;94℃变性1 min,52℃退火45 s,72℃延伸1 min,30次循环;72℃延伸8 min。对测序结果分析表明:扩增产物的大小为200~2 000 bp,23份材料与对照材料存在单碱基的缺失,利用单碱基变异可以区分出24份材料,说明18份优良营养系材料发生了遗传变异。【结论】SCoT分子标记可用于梨树营养系变异的鉴定。
[Objective] The ＇Kuerle Xiangli＇ （Pyrus sinkiangensis Yti） is one of the high quality and local products in Xinjiang. The fruits have good yellow-green skin, fine texture and endurable storage, and are crispy, tender, juicy and sweet. The predecessors have done lots of research works on Pyrus such as efficient cultivation, botanical classification, gene cloning and expression analysis and identification of variety. Molecular markers have also been employed in Pyrus for agronomic characters except for SCoT mark- er. The most significant advantage of SCoT molecular marker is commonality between species. We aimed to establish a SCoT-PCR system with good stability, repeatability, and polymorphism for pear plants and to use it to identify the bud sports in ＇ Kuerle Xiangli＇, [ Methods ] The sports of ＇ Kuerle Xiangli＇ were collected in Xinjiang province. SCoT-PCR systems were screened and the amplification reaction procedures were optimized by orthogonal experimental design L25 （56）. The genomic DNA of twenty-four suspected mutants of ＇Kuerle Xiangli＇ were amplified with two primers which randomly selected from twenty prim- ers. The PCR products were recycled by TIANGEN DNA gel extraction Kit. The carrier pMD19-T was used to connect the products with state DH5ot. The regular bacterium solution was used to extract recombi- nant plasmid. The recombinant plasmid was sequenced in BGI. After sequencing, proofread and spliced the sequence. The potential ORFs were found by Editseq and ORF Finder. The nucleotide sequences were analyzed with multiple comparison by DNAMAN. The DNAMAN outputted the Homology matrix and distance matrix. The phylogenetic tree was outputted by MEGA 5.0. [Results] The electrophoresis resuits of PCR productws showed that the bands were clear and the polymorphisms were good. The order of various factors＇ influence of PCR reaction system was dNTPs〉 Taq enzyme〉 primer〉 Mg2 ＋〉 template DNA. The biggest factor to affect ＇Kuerle Xiangli＇ SCoT-PCR reaction system was the concentration of dNTPs. The results of variance analysis showed that the impacts of various factors on SCoT-PCR amplification results were nor significant. Therefore, we determined the best combination of PCR system directly in accordance with the PCR products＇ electrophoresis results. The optimal PCR system was in total volume of 20 p,L contained 10xbuffer with Mg2＋ 2.5 μL, 1.5 U Taq polymerase, 10 μmol· L-1 primer, 25 ng template DNA, and 1.6 mmol· L^-1 dNTPs, and reaction procedure was： 94 ℃ for 1 rain, followed by 30 cycles of 94 ℃ for 1 min, 52 ℃ annealing temperature for 45 s, 72℃ for 1 min, and then terminated with a 8 min extension step at 72 ℃. We had screened the 20 available primers from Collard＇ s primers. The sequencing results showed that amplified products sizes ranged from 200 bp to 2 000 bp and the stripes of amplification were distinctive. There were single-base＇ s deficiency among 23 suspected mutants compared with the control. Single-base mutation could be used to distinguish 24 materials. We inferred that 18 of 23 suspected mutants were bud mutants. [Conclusion] An optimized SCoT-PCR reaction system was suitable for ＇Kuerle Xiangli＇. SCoT molecular markers could be used for identification of sports in pear plant.
出处
《果树学报》
CAS
CSCD
北大核心
2016年第11期1337-1346,共10页
Journal of Fruit Science
基金
兵团"科技人员服务南疆专项"(2010GG59)
兵团科技攻关计划(2011BA003-3)
