伊犁郁金香cpDNA-PCR体系构建与遗传多样性分析  

Establishment of cpDNA-PCR reaction system and genetic diversity analysis of Tulipa iliensis

作　　者：秦斗文 刘伟强 田吉婷 巨秀婷 QIN Douwen;LIU Weiqiang;TIAN Jiting;JU Xiuting(College of Agriculture and Animal Husbandry,Qinghai University,Xining 810016,China;Key Laboratory of Landscape Plants and Ornamental Horticulture of Qinghai Province,Xining 810016,China)

机构地区：[1]青海大学农牧学院,青海西宁810016 [2]青海省园林植物与观赏园艺重点实验室,青海西宁810016

出　　处：《浙江农业学报》2025年第1期78-89,共12页Acta Agriculturae Zhejiangensis

基　　金：2021年度“西部之光”人才培养计划(中科人字〔2022〕4号)。

摘　　要：为明确伊犁郁金香(Tulipa iliensis)的遗传背景,在叶绿体基因组水平开展遗传多样性研究。本研究以伊犁郁金香为研究对象,对cpDNA-PCR反应体系中的Mg^(2+)、dNTPs、引物浓度、Taq DNA酶和模板DNA浓度在L16(45)正交试验设计的基础上结合单因素优化筛选试验构建伊犁郁金香cpDNA-PCR反应体系,在最优体系的基础上进行引物筛选并通过引物筛选完成cpDNA-PCR扩增,以验证cpDNA-PCR反应体系的稳定性,并对伊犁郁金香野生群体进行遗传多样性分析。结果表明,构建的伊犁郁金香最佳cpDNA-PCR反应体系(25μL)为Mg^(2+)2.00 mmol·L^(-1),dNTPs 0.20 mmol·L^(-1),引物浓度0.40μmol·L^(-1),Taq DNA酶0.50 U,模板DNA浓度110 ng。利用最佳扩增体系,从16对候选引物组合中筛选出可用于后续伊犁郁金香cpDNA-PCR扩增的14对引物(trnK-rps16,M3-M4,F71-R1516,trnD-trnE,psbB-psbH,trnL-trnF,atpB-rbcL,a1-b1,1F-1R,rps8-rp116,atpI-atpH,accD-psaI,petG-trnP,2F-2R)。建立的伊犁郁金香cpDNA-PCR反应体系扩增稳定,条带清晰,对54个伊犁郁金香个体进行遗传多样性分析,引物psbB-psbH扩增片段平均长度为582 bp,其中多态性位点数284个,核苷酸多态性Pi为0.055、平均核苷酸差异数K为30.261,基因流(Nm=1.21>1),表明伊犁郁金香居群遗传多样性高,不同居群间基因交流频繁。该研究建立的cpDNA-PCR反应体系可用于伊犁郁金香遗传多样性和种群遗传结构研究,研究结果可以为野生郁金香种质资源的合理保护、可持续利用提供理论依据和技术支撑,为科学保护伊犁郁金香种质资源提供依据。To clarify the genetic background of Tulipa iliensis,the genetic diversity was studied at the level of chloroplast genome.The cpDNA-PCR reaction system of T.iliensis was constructed by using the L16(45)orthogonal experiment design and single factor optimization screening experiment.The system was optimized by adjusting the Mg^(2+),dNTPs,primer concentration,Taq DNA polymerase,and template DNA concentration in the cpDNA-PCR reaction system.With the optimal system,primers were screened and performed cpDNA-PCR amplification to verify the stability of the reaction system,and the genetic diversity of the wild population of T.iliensis was also analyzed.The results showed that the optimal cpDNA-PCR reaction system for T.iliensis consisted of 2.00 mmol·L^(-1) Mg^(2+),0.20 mmol·L^(-1) dNTPs,0.40μmol·L^(-1) primer concentration,0.50 U Taq DNA polymerase,and 110 ng template DNA concentration.Using the optimal amplification system,14 pairs of candidate primers(trnK-rps16,M3-M4,F71-R1516,trnD-trnE,psbB-psbH,trnL-trnF,atpB-rbcL,a1-b1,1F-1R,rps8-rp116,atpI-atpH,accD-psaI,petG-trnP,2F-2R)were selected from 16 pairs of candidate primer combinations for subsequent cpDNA-PCR amplification of T.iliensis.The established cpDNA-PCR reaction system for T.iliensis was stable in amplification and produced clear bands,genetic diversity analysis was performed on 54 T.iliensis individuals,The average length of amplified fragments using primer psbB-psbH was 582 bp,with 284 identified polymorphic sites,the nucleotide polymorphism Pi was 0.055,the average nucleotide difference number K was 30.261,and the gene flow(Nm=1.21>1)indicated that the genetic diversity of the T.iliensis population was high,and gene exchange among different populations was frequent.The cpDNA-PCR reaction system established in this study can be used to study the genetic diversity and population genetic structure of T.iliensis,providing a theoretical basis and technical support for the rational protection and sustainable utilization of wild tulip germplasm resources,an

关 键 词：伊犁郁金香 CPDNA 体系构建 遗传多样性 

分 类 号：S682.2.63[农业科学—观赏园艺]