An Improved Chloroplast DNA Extraction Procedure for Whole Plastid Genome Sequencing 英文参考文献.docVIP
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An Improved Chloroplast DNA Extraction Procedure for Whole Plastid Genome Sequencing 英文参考文献
AnImprovedChloroplastDNAExtractionProcedurefor
WholePlastidGenomeSequencing
ChaoShi1,2.,NaHu1,2.,HuiHuang1,JuGao1,You-JieZhao1,Li-ZhiGao1*
1Plant Germplasm and Genomics Center, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, China, 2Graduate School of Chinese Academy of
Sciences,Beijing,China
Abstract
Background:Chloroplastgenomessupplyvaluablegeneticinformationforevolutionaryandfunctionalstudiesinplants.
Thepastfiveyearshavewitnessedadramaticincreaseinthenumberofcompletelysequencedchloroplastgenomeswith
the application of second-generation sequencing technology in plastid genome sequencing projects. However, cost-
effectivehigh-throughputchloroplastDNA(cpDNA)extractionbecomesamajorbottleneckrestrictingtheapplication,as
conventionalmethodsaredifficulttomakeabalancebetweenthequalityandyieldofcpDNAs.
Methodology/PrincipalFindings:WefirsttestedtwotraditionalmethodstoisolatecpDNAfromthethreespecies,Oryza
brachyantha,LeersiajaponicaandPrinsepiautihis.BothofthemfailedtoobtainproperlydefinedcpDNAbands.However,
we developed a simple but efficient method based on sucrose gradients and found that the modified protocol worked
efficientlytoisolatethecpDNAfromthesamethreeplantspecies.WesequencedtheisolatedDNAsampleswithIllumina
(Solexa) sequencing technology to test cpDNA purity according to aligningsequence reads to the reference chloroplast
genomes,showingthatthereferencegenomewasproperlycovered.Weshowthat40–50%cpDNApurityisachievedwith
ourmethod.
Conclusion: Here we provide an improved method used to isolate cpDNA from angiosperms. The Illumina sequencing
results suggest that the isolated cpDNA has reached enough yield andsufficient purity to perform subsequent genome
assembly. The cpDNA isolation protocol thus will be widely applicable to the plant chloroplast genome sequencing
projects.
Citation:ShiC,HuN,HuangH,GaoJ,ZhaoY-J,etal.(2012)AnImprovedChloroplastDNAExtractionProcedureforWholePlastidGenomeSequencing.PLoS
ONE7(2):e31468.doi:10.1371/journal.pone.
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