Abstract
BackgroundThe 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.ResultsWe 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.ConclusionsAs 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s13007-016-0139-1) contains supplementary material, which is available to authorized users.
Highlights
The double digest restriction-site associated DNA sequencing technology is a reduced representation sequencing technology by sampling genome-wide enzyme loci developed on the basis of nextgeneration sequencing. double digest restriction associated DNA (ddRAD)-seq has been widely applied to single nucleotide polymorphism (SNPs) marker development and genotyping on animals, especially on marine animals as the original ddRAD protocol is mainly built and trained based on animal data
RAD series mainly includes IIB restriction endonucleases restriction-site associ‐ ated DNA (2b-RAD) [21], ddRAD [22] and ezRAD [23]. 2b-RAD adopts a kind of type II restriction endonuclease to digest the genome, producing only ~33 bp fragments, which lack of biases due to fragment size selection but may restrict the potential for discovering more SNPs. ezRAD is the only protocol that relies on illumina authoritative kits to construct the library with customer support but the cost is still not as low as the author claimed [24]. ddRAD can tune fragments number by employing two different enzymes and size selection, and the process of constructing a library is quite simple while genomic DNA it requires is of the highest quality in all the RAD methods [24]
To validate the genotyping accuracy, we presented linkage map results of one D. latiflorus F1 mapping population (Guoqian Yang et al, unpublished data) according to modified double digest restriction associated DNA (MiddRAD) protocol and 55 genotypes were randomly selected and verified by independent Sanger sequencing
Summary
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 nextgeneration 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. 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 nextgeneration sequencing. Restriction-site associated DNA sequencing technology (RAD-seq) is a reduced representation sequencing technology by sampling genome-wide single enzyme loci developed on the basis of next-generation sequencing [1, 2]. The technology breaks genome into a certain size of DNA fragments by employing a restriction endonuclease (usually a low-frequency cutter) combined with the ultrasonic shearing method, the fragmented DNA is enriched for constructing a sequencing library so that sequences beside the cleavage site can be acquired for high-throughput sequencing [3]. DdRAD can tune fragments number by employing two different enzymes and size selection, and the process of constructing a library is quite simple while genomic DNA it requires is of the highest quality in all the RAD methods [24] RAD series mainly includes 2b-RAD [21], ddRAD [22] and ezRAD [23]. 2b-RAD adopts a kind of type II restriction endonuclease to digest the genome, producing only ~33 bp fragments, which lack of biases due to fragment size selection but may restrict the potential for discovering more SNPs. ezRAD is the only protocol that relies on illumina authoritative kits to construct the library with customer support but the cost is still not as low as the author claimed [24]. ddRAD can tune fragments number by employing two different enzymes and size selection, and the process of constructing a library is quite simple while genomic DNA it requires is of the highest quality in all the RAD methods [24]
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