title: Development of a universal and simplified ddRAD library preparation approach for SNP discovery and genotyping in angiosperm plants.
abstract: 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.
authors: Germplasm Bank of Wild Species, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, 650201 China ; Kunming College of Life Sciences, University of Chinese Academy of Sciences, Kunming, 650201 China.
authors: Guo-Qian
authors: GQ
authors: Yang
authors: Germplasm Bank of Wild Species, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, 650201 China ; Kunming College of Life Sciences, University of Chinese Academy of Sciences, Kunming, 650201 China.
authors: Yun-Mei
authors: YM
authors: Chen
authors: Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao, 266071 China.
authors: Jin-Peng
authors: JP
authors: Wang
authors: Germplasm Bank of Wild Species, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, 650201 China ; Kunming College of Life Sciences, University of Chinese Academy of Sciences, Kunming, 650201 China.
authors: Cen
authors: C
authors: Guo
authors: Germplasm Bank of Wild Species, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, 650201 China ; Kunming College of Life Sciences, University of Chinese Academy of Sciences, Kunming, 650201 China.
authors: Lei
authors: L
authors: Zhao
authors: Germplasm Bank of Wild Species, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, 650201 China.
authors: Xiao-Yan
authors: XY
authors: Wang
authors: Germplasm Bank of Wild Species, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, 650201 China ; Kunming College of Life Sciences, University of Chinese Academy of Sciences, Kunming, 650201 China.
authors: Ying
authors: Y
authors: Guo
authors: Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao, 266071 China.
authors: Li
authors: L
authors: Li
authors: Germplasm Bank of Wild Species, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, 650201 China.
authors: De-Zhu
authors: DZ
authors: Li
authors: Germplasm Bank of Wild Species, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, 650201 China.
authors: Zhen-Hua
authors: 0000-0002-5040-3137
authors: ZH
authors: Guo
keywords: Genotype-by-sequencing; MiddRAD; Next-generation sequencing; RAD-seq; ddRAD