基干ITS条形码序列对枸杞杂交种早期鉴定.docVIP

基于ITS条形码序列对枸杞杂交种的早期鉴定 　　摘要：采用改进CTAB法提取枸杞（Lycium Linn）叶片DNA，利用合成的特异引物对其DNA中nrDNA ITS区进行扩增、克隆，对目的片段进行测序分析，以期利用ITS条形码序列对枸杞杂交种进行早期鉴定。结果表明，7份枸杞属不同种间杂交种的ITS序列长度变异范围为558～632 bp，平均为610 bp。排序后的总长度为632 bp，整个转录间隔区（ITS1+ITS2）对位排列后总长度为478 bp，共有138个变异位点，ITS1和ITS2分别为85和53个，占28.9%；保守位点340个，占71.1%；有6个信息位点，占1.3%；51个转换位点，12个颠换位点，其中ITS1区的信息位点所占比例低于ITS2区，而ITS1区的转换位点与颠换位点比值高于ITS2区。通过对宁夏枸杞、北方枸杞、黑果枸杞及其种间杂交育种产生的杂交后代分析，得出基于ITS聚类分析能够初步判别杂交后代与父母本的亲缘关系与差异，可以作为早期鉴定枸杞属不同种间杂交种后代的方法之一 关键词：枸杞（Lycium Linn）；ITS序列；鉴定；杂交育种 中图分类号：S567.1+9 文献标识码：A 文章编号：0439-8114（2016）19-5075-03 DOI：10.14088/j.cnki.issn0439-8114.2016.19.045 Abstract：To identify Lycium Linn hybrids in early time by the nrDNA ITS sequence，using improved CTAB method to acquire DNA of Lycium Linn leaf， nrDNA ITS area of DNA was propagated and cloned by composite special primers，and sequence of the target segment was analyzed. The results show that the length variation range of nrDNA ITS sequence is 558～632 bp and the average is 610 bp. The length is 632 bp after sorting and the total length of ITS（ITS1+ITS2） is 478 bp by alignment，including 138 variation sites（85 variation sites in ITS1 and 53 variation sites in ITS2） which occupied 28.9%，340 conserved sites accounted for 71.1%. There are 6 information sites， 51 transformation sites and 12 transversion sites totally，but the proportion of information sites in ITS1 is lower than in ITS2，and the ratio of transformation sites and transversion sites in ITS1 is higher than in ITS2. According to analysis of L. ruthenicum Murr.，L. barbarum Linn.，L. chinense and the hybrids crossbred among these species，we could judge the genetic relationship and difference between hybrids and their parents based on nrDNA ITS sequence. In concluding，the nrDNA ITS sequence analysis is one way to identify Lycium crossbreeding among species in early time. Key words： Lycium Linn； internal transcribed spacer； identification； crossbreeding 枸杞（Lycium Linn）富含枸杞多糖、甜菜碱、类胡萝卜素、多种不饱和脂肪酸等多种有效成分，具有抗氧化、抗肿瘤、延缓衰老、增强免疫力、