快速鉴定紫花苜蓿混入苜蓿菟丝子的分子方法及其通用性

A universal molecular method for rapid identification of alfalfa and dodder seeds

紫花苜蓿(Medicago sativa)种子和杂草菟丝子(Cuscuta spp.)种子在颜色、大小和形状上有很多相似之处。传统牧草种子纯度的评价方法是形态学比较,主要依赖感官辨认和经验,但是,种子形态间的相似性极大增加了形态鉴别的难度,耗时长且不准确。本研究基于紫花苜蓿和寄生植物苜蓿菟丝子(C.approximata)叶绿体rbcL(ribulose-1,5-bisphosphate carboxylase/oxygenase large,rbcL)基因的序列差异,设计针对苜蓿菟丝子的特有引物,对紫花苜蓿和苜蓿菟丝子的DNA同时进行PCR扩增,最终通过观察是否扩增出苜蓿菟丝子的DNA片段,来检验紫花苜蓿的种子纯度。此外,在苜蓿菟丝子和紫花苜蓿种子DNA和种子粒数比例分别为1꞉10000和1꞉1000时,该方法也可以准确扩增出紫花苜蓿中混杂的菟丝子。因此,该方法具有可靠性高、检测速度快、灵敏度高等优点,为海关检疫紫花苜蓿种子中是否携带寄生植物苜蓿菟丝子种子提供便捷的检测技术,对种子生产和牧草育种具有重要意义。

There are numerous similarities between Medicago sativa seeds and those of Cuscuta spp.with respect to color,size,and shape.Traditionally,the evaluation of seed purity has been based primarily on morphological methods,which are mainly dependent on visual assessments.However,given that the morphological indices of similar forage seeds are often similar or overlapping,the consistency of replicated seed observations is unreliable,and the process is laborious and timeconsuming.By way of resolving these problems,in this study,we investigated the utility of a molecular approach based on sequence differences between the chloroplast rbcL(ribulose-1,5-bisphosphate carboxylase/oxygenase large)genes of alfalfa and parasitic dodder,using dodder-specific primers designed for PCR amplification of alfalfa and dodder DNA.In this approach,the purity of alfalfa seeds was assessed by observing whether fragments of dodder DNA were amplified.In addition,this method can also accurately amplify DNA from mixtures of dodder seeds in alfalfa when the proportions of dodder to alfalfa seed DNA and seed number were 1꞉10000 and 1꞉1000,respectively.Thus,given its reliability,rapidity,and sensitivity,this method can provide a convenient detection technology for customs quarantine,thereby enabling assessments of alfalfa seed purity.Moreover,it has considerable potential for extensive application in the seed production and breeding of forage crops.