[1]刘舒宇,刘 霞,李金花*,等.杨柳科单拷贝核基因引物的开发与利用[J].南京林业大学学报(自然科学版),2019,43(05):044-50.[doi:10.3969/j.issn.1000-2006.201811015]
 LIU Shuyu,LIU Xia,LI Jinhua*,et al.Development of single copy nuclear gene primers for the family Salicaceae[J].Journal of Nanjing Forestry University(Natural Science Edition),2019,43(05):044-50.[doi:10.3969/j.issn.1000-2006.201811015]





Development of single copy nuclear gene primers for the family Salicaceae
刘舒宇1刘 霞2李金花1*王兆山1*
(1.中国林业科学研究院林业研究所,北京 100091; 2.重庆文理学院特色植物研究院,重庆 402160)
LIU Shuyu1 LIU Xia2 LI Jinhua1* WANG Zhaoshan1*
(1. Research Institute of Forestry, Chinese Academy of Forestry, Beijing 100091, China; 2. Institute of Special Plants, Chongqing University of Arts and Sciences, Chongqing 402160, China)
杨柳科 单拷贝核基因 多态性 系统发育关系
Salicaceae single-copy nuclear gene polymorphism phylogenetic relationship
【目的】由于现有的遗传标记比较单一且变异太少,杨柳科(Salicaceae)属间系统发育关系还存在较大的争议,需要开发在种间变异大且信息量多的标记来准确揭示杨柳科属间系统位置。相比胞质基因组,单拷贝核基因具有双亲遗传、携带大量信息位点和直系同源的特点,可以满足不同系统发育问题及不同分类阶元研究的需要。【方法】根据先前研究中发表的15个杨柳科(杨属、柳属)单拷贝核基因标记以及核糖体DNA的转录间隔区序列(ITS),选取杨柳科4个属的代表物种:箣柊(Scolopia chinensis)、大果刺篱木(Flacourtia ramontchi)、爪哇脚骨脆(Casearia velutina)、天料木(Homalium cochinchinense)进行PCR扩增测序,以测序结果为参考序列,设计合适的引物。利用杨柳科6个不同属的代表物种:山桂花(Bennettiodendron leprosipes)、柞木(Xylosma congesta)、锡兰莓(Dovyalis hebecarpa )、栀子皮(Itoa orientalis)、山桐子(Idesia polycarpa)、山拐枣(Poliothyrsis sinensis)来验证引物的通用性。利用箣柊一个自然居群中的20个个体对新引物进行多态性计算以及中性检验。将单拷贝核基因序列和ITS序列组合成联合片段,分别用最大简约法和贝叶斯法构建系统发育树。【结果】在10个属中成功筛选设计出6对合适的引物,分别为5对单拷贝核基因标记以及ITS遗传标记,各引物扩增出的序列长度为297~716 bp。多态性检测结果表明:单倍型数目(H)为3~9个,平均核苷酸差异数(κ)为0.644~2.278,核苷酸多样性(π)和Watterson核苷酸多样性参数(θw)分别为0.001 04~0.004 51和0.001 19~0.004 75,所有位点在箣柊中核苷酸多样性较高。中性检验结果表明所有位点均符合中性进化假设。利用位点联合方法构建的最大简约树和贝叶斯树的拓扑结构基本一致,各分支均有较高的支持率。【结论】筛选出的6对通用性高的引物,丰富了杨柳科的分子标记,可为杨柳科属间系统发育学以及杨柳科植物遗传多样性的研究提供参考。
【Objective】The phylogenetic relationships among genera in the Salicaceae family have remained controversial, largely because the available genetic markers lack polymorphisms. More efficient and highly variable genetic markers are needed to address lingering questions about taxonomic relationships within the Salicaceae family. Compared with the cytoplasmic genome, single-copy nuclear genes(SCNGs)are inherited bi-parentally, carry a large number of information sites, and are orthologous, and therefore can better reflect phylogenetic relationships among species and satisfy the requirements of different taxa. 【Method】Based on the 15 SCNG markers of old Salicaceae members(Populus and Salix)and the internal transcribed spacer(ITS)sequences published in previous studies, PCR amplification and sequencing were carried out in 4 species in the Salicaceae family: Scolopia chinensis, Flacourtia ramontchi, Casearia velutina and Homalium cochinchinense. The primers were designed based on the sequencing results. Then, we used the representative species of 6 different genera of Salicaceae(Bennettiodendron leprosipes, Xylosma congesta, Dovyalis hebecarpa, Itoa orientalis, Idesia polycarpa and Poliothyrsis sinensis)to verify the application of the primers. Polymorphism and neutral tests were performed on the new primers using 20 individuals in a population of S. chinensis. Maximum parsimony and Bayesian inference methods were used to construct the phylogenetic trees by combining data(SCNGs+ITS). 【Result】Six pairs of primers(5 SCNG markers and ITS)with an aligned length ranging from 297 to 716 bp were successfully identified from 10 genera. The results of polymorphism analysis of the 6 primers showed that the number of haplotypes(H)ranged from 3 to 9, the average nucleotide difference(κ)was 0.644-2.278, the nucleotide diversity(π)and the Watterson's parameter(θw)were 0.001 04-0.004 51 and 0.001 19-0.004 75, respectively. Neutral test results indicated that all loci were consistent with neutral evolutionary assumptions. Two phylogenetic trees showed similar topology and both could be classified well with high support values based on combined data. 【Conclusion】The six markers developed in this study are anticipated to enrich molecular markers for species in the Salicaceae family, and provide useful information for elucidating the phylogenetic relationships and genetic diversity within Salicaceae.


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收稿日期:2018-11-06 修回日期:2019-05-05 基金项目:国家自然科学基金面上项目(31470665)。 第一作者:刘舒宇(1534908913@qq.com)。*通信作者:李金花(lijinh@caf.ac.cn),副研究员,负责论文修改,ORCID(0000-0001-5056-6692); 王兆山(w@caf.ac.cn),副研究员,负责实验设计及实验指导,ORCID(0000-0002-8221-1420)。 引文格式:刘舒宇,刘霞,李金花,等. 杨柳科单拷贝核基因引物的开发与利用[J]. 南京林业大学学报(自然科学版),2019,43(5):44-50.
更新日期/Last Update: 2019-10-08