Optimization of start codon targeted polymorphism PCR system and identification of bud mutant in ‘Kuerle Xiangli'

Abstract:【Objective】The‘Kuerle Xiangli'(Pyrus sinkiangensis Yü) 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 marker. 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 L_(25)(5~6). The genomic DNA of twenty-four suspected mutants of‘Kuerle Xiangli'were amplified with two primers which randomly selected from twenty primers. The PCR products were recycled by TIANGEN DNA gel extraction Kit. The carrier p MD19-T was used to connect the products with state DH5α. The regular bacterium solution was used to extract recombinant 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 results 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 d NTPs> Taq enzyme> primer> Mg2 +> template DNA. The biggest factor to affect‘Kuerle Xiangli'SCoT-PCR reaction system was the concentration of d NTPs. The results of variance analysis showed that the impacts of various factors on SCoT-PCR amplification results were not 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 μL contained 10×buffer 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 d NTPs, and reaction procedure was: 94 ℃ for 1 min, followed by 30 cycles of 94 ℃ for 1 min, 52 ℃ annealing temperature for 45 s, 72 ℃ for 1 min, and then terminated with a 8min 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 that18 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.