基于rbcL-a和ITS的枸杞鉴别、遗传关系分析及ITS假基因的发现

Identification,Phylogenetic Relationship Analysis of Lycium Based on rbcL-a and ITS Sequence and the Discovery of ITS Pseudogene

利用rbcL-a及ITS序列作为分子标记,对14个枸杞种或品种及1个伪品白刺进行了真伪鉴别及遗传关系分析。CTAB法提取总DNA,用通用引物对rbcL-a和ITS序列扩增、克隆、测序及分析,T克隆解决部分样品ITS无法成功测序问题。rbcL-a和ITS序列通用引物可在所有样品中成功扩增,rbcL-a序列全长643 bp,在14个枸杞种或品种中完全一致,与白刺具有45bp(7%)的碱基变异。ITS序列在14个枸杞种或品种中长度变异范围为513-692 bp。采用UPGMA法构建系统树,可区分白刺与枸杞,黑果枸杞和宁夏枸杞各品种分别聚为两支,云南枸杞、韩国枸杞及黄果枸杞则聚在宁夏枸杞大分支内。rbcL-a序列可有效进行枸杞真伪鉴别,ITS序列可用于枸杞品种鉴定及遗传关系分析。此外,本研究首次揭示了枸杞品种及个体内的ITS多态性,发现在枸杞中存在ITS假基因这一现象,为未来利用ITS序列进行枸杞系统进化研究提供一些新的参考。

Identification and phylogenetic relationship analysis of 14 species or cultivars of Lycium and 1 adulterant of Nitraria tangutorum were studied using rbcL-a and ITS as molecular markers. Total DNA was extracted by CTAB method. With universal primers, rbcL-a and ITS were amplified, cloned, sequenced and analyzed via PCR. T cloning technique was used to deal with the unsuccessful sequencing of ITS sequence of some samples, rbcL-a and ITS sequences with universal primers were amplified successfully in all samples. The lengths of rbcL-a in Lycium and N. tangutorum were 643 bp, and the sequences of rbcL-a were the same in all samples of Lycium, but existed 45 bp variation sites （ 7% ） compared with IV. tangutorum. The lengths of ITS in 14 species or euhivars of Lyeium ranged from 513 - 692 bp. The UPGMA phylogenetie tree was constructed based on ITS sequence to distinguish samples of Lycium and N. tangutorum well. ITS sequences of L ruthenicum and L barbarura were clustered into two groups, respectively. L. yunnanense, IL chinense and L. barbarum var. a.uranticarpum were classed as the same branch of L. barbarum. The marker of rbcL-a can effectively identify Lycium from its adulterant. The molecular marker of ITS is applicable in variety identification and.genetic relationship analysis of Lycium. This study revealed the polymorphism of ITS in L barbarum L. varieties and individuals for the first time, and the presence of ITS pseudogenes in L barbarum L., which offers some new views for systematic evolution research of Lycium using ITS sequence.