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      1. 我国学者改进简化基因测序方法 有效简化基因建库仪器配置
        昆明植物研究所 · 2016/08/10
        该技术操作简单灵活、检测成本低、检测通量高,更易被科研人员掌握并能在通用分子实验室中实现,特别适用于需要对大量个体进行SNP标记开发及分型的遗传图谱构建、?#20302;?#21457;育分析及群体遗传学等研究


        在二代测序基础上发展起来的RAD-seq技术是一项基于全基因组酶切位点的简化基因组测序技术。由于特异性酶切位点在全基因组范围内广泛分布,通过RAD-seq能够在大多数物种内获得数万至数十万的单核苷酸多态性(single nucleotide polymorphism,SNP)标记。

        在此基础上,RAD-seq技术又衍生出多种简化基因组测序方法,包含GBS,ddRAD,ezRAD以及2b-RAD等。其中,ddRAD-seq通过一个稀有酶与一个常见酶相结合对基因组DNA进行双酶切,免去随机打断的过程,是一种非常有前景的简化基因组测序技术。

        不过,ddRAD技术虽然在建库流程上比RAD做了一定程度的简化,但仍然包括12步,实验耗时较长。因此,有必要对现有的ddRAD简化基因组测序文库构建方法进行改进,以克服其需要多次选酶、使用仪器复杂、成本偏高等缺陷,提高测序效率。

        中国科学院昆明植物研究所博士研究生杨国骞在研究员郭振华与李德铢指导下,与中科院海洋研究所李莉研究组一起,开发出一种被子植物中通用的双酶切简化基因组(Modified ddRAD,简称MiddRAD)二代测序文库构建方法。

        该方法首先发现一种被子植物通用酶切组?#31232;癆vaII + MspI?#20445;?#20943;少了不同物种间需要多次选酶的步骤;其次,该方法将复杂的建库专用仪器优化为通用的分子生物学仪器;再次,该方法将P1测序接头由37个碱基简化为25个碱基(barcode按5个碱基计算)并设计出一套新的barcode-adapter体系;最后,该方法还减少了纯化酶切产物、连接前定量及混样后纯化连接产物的步骤,简化了建库流程,允许仅使用50ng DNA?#32431;?#23436;成文库构建。

        该技术操作简单灵活、检测成本低、检测通量高,更易被科研人员掌握并能在通用分子实验室中实现,特别适用于需要对大量个体进行SNP标记开发及分型的遗传图谱构建、?#20302;?#21457;育分析及群体遗传学等研究。因此,MiddRAD-seq在农业分子育种、进化生物学和保护生物学等领域具有良好的应用价值和推广前景。

        研究以Development of a universal and simplified ddRAD library preparation approach for SNP discovery and genotyping in angiosperm plants 为题发表于Plant Methods上。

        该研究得到国?#26131;?#28982;科学基金项目(31470322、31430011)的支持。


        MiddRAD(a)与ddRAD(b)实验流程图

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        • Development of a universal and simplified ddRAD library preparation approach for SNP discovery and genotyping in angiosperm plants

          Background The double digest restriction-site associated DNA sequencing technology (ddRAD-seq) is a reduced representation sequencing technology by sampling genome-wide enzyme loci developed on the basis of next-generation sequencing. ddRAD-seq has been widely applied to SNP marker development and genotyping on animals, especially on marine animals as the original ddRAD protocol is mainly built and trained based on animal data. However, wide application of ddRAD-seq technology in plant species has not been achieved so far. Here, we aim to develop an optimized ddRAD library preparation protocol be accessible to most angiosperm plant species without much startup pre-experiment and costs. Results We first tested several combinations of enzymes by in silico analysis of 23 plant species covering 17 families of angiosperm and 1 family of bryophyta and found AvaII + MspI enzyme pair produced consistently higher number of fragments in a broad range of plant species. Then we removed two purifying and one quantifying steps of the original protocol, replaced expensive consumables and apparatuses by conventional experimental apparatuses. Besides, we shortened P1 adapter from 37 to 25 bp and designed a new barcode-adapter system containing 20 pairs of barcodes of varying length. This is an optimized ddRAD strategy for angiosperm plants that is economical, time-saving and requires little technical expertise or investment in laboratory equipment. We refer to this simplified protocol as MiddRAD and we demonstrated the utility and flexibility of our approach by resolving phylogenetic relationships of two genera of woody bamboos (Dendrocalamus and Phyllostachys). Overall our results provide empirical evidence for using this method on different model and non-model plants to produce consistent data. Conclusions As MiddRAD adopts an enzyme pair that works for a broad range of angiosperm plants, simplifies library constructing procedure and requires less DNA input, it will greatly facilitate designing a ddRAD project. Our optimization of this method may make ddRAD be widely used in fields of plant population genetics, phylogenetics, phylogeography and molecular breeding.

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