AFSM Sequencing:Development And Application Of A Simple And Rapid Method For Genome-wide SNP And Methylation Site Discovery

We developed AFSM(amplified fragments SNP and methylation)sequencing approach,based on the use of a next-generation sequencing platform and two restriction enzyme paris Eco RI-Msp I and Eco RI-Hpa II.Two sets of 96 adapter pairs were developed to concurrently identify SNPs,indels and Methylation sites for 96 lines in population.AFSM is particularly suitable for high-throughput full methylation and semi-methylation analysis.Selective amplification of the genome using restriction enzymes reduces genome complexity.At the same time,it can increase the number of samples tested,reduce the sequencing coverage of individual samples,and greatly reduce the cost of resequencing.Can be applied to any species and is highly reproducible.Because the sequence of the tag structure minimizes the cost of reagents in the library.The location of the tag,upstream of the cleavage site of the genomic DNA.At the same time,a minimum of 96 samples were detected and genotyping was completed.Using Hiseq2500 sequencing,the cost of resequencing is reduced on a large scale,achieving efficiency and cost effectiveness.Selecting the Msp Ⅰ/Hpa Ⅱ(CCGG)restriction site can increase the probability of ORF regions in the captured genome.SNPs in these gene regions are more valuable for genome-wide association analysis.It also circumvents some genomic repeat regions,simplifying challenging assembly problems and high genetic diversity levels for species.Hybrid double digestion is used to generate DNA methylation polymorphism sites with sequence information based on differences in DNA methylation sensitivity.It is not necessary to know the sequence information of the DNA to be tested,and it is versatile in different organisms,and can be used for organisms whose background knowledge of DNA sequences is unknown.The procedure is relatively simple and can detect cytosine methylation changes at the CCGG site in a genome-wide range.To further demonstrate the reliability and practicability of this approach,we used AFSM approach in genetic linkage map construction in Jatropha curcas,QTL mapping in Hevea brasiliensis and the DNA methylatic variation across Aegilops tauschii nature poputation to response to climate conditions and local adaptation.1.The construction of genetic linkage map of Jatropha curcas.As a sustainable and renewable energy source,bioenergy,Jatropha curcas characterized by high oil content,is one of the highest potential energy plants among oil-bearing tree species.Genetic linkage map is an important tool for genetic analysis and molecular breeding.However,the lack of sufficient polymorphism markers has limited the molecular breeding in Jatropha curcas.Therefore,we designed and developed AFSM approach.Using 153 lines in Jatropha curcas genetic population,the first high-density genetic linkage map was constructed in Jatropha curcas,contained 3422 SNP and indel markers,with an average mark density of 0.403 c M,and 1380.58 c M in total length.2.Construction of an ultra-high density genetic linkage map for Hevea brasiliensis and identification of QTL for dry latex yield.Hevea brasiliensis is the most important commercial producer of natural rubber,because of its advantages of high yield and good quality rubber production.In this study,571267 SNP and 134184 indel markers were identified,in 187 lines from rubber tree population derived from a cross between a female parent Yunyan277-5 and a male parent IAN873.We constructed an ultra-high density genetic linkage map contained 6940 SNP and indel markers,with average marker density and recombination rate 0.30 c M and 0.97 c M/Mb,respectively.Then the whole genome QTL scanning for dry latex yield trait were performed and seventeen rep QTLs were obtained,among which q DFY-10 and q DFY-18-4 could explain up to 38.3% and33.3% phenotypic variability,respectively.Numerous highly-promising QTL candidate genes were identified and then verified significant associated with DLY trait,including some first found in rubber tree,such as thioredoxin h,plastin-like protein,calmodulin binding protein,cytochrome-c oxidase and methylglutaconyl-Co A hydratase.3.Analysis of genomic DNA methylation and global environmental adaptability in Aegilops tauschii.Aegilops tauschii,one of gramineous species,is the diploid ancestor of the D genome of hexaploid wheat(Triticum aestivum),with genetically diverse.It has been widely accepted that A.tauschii ssp.strangulata is the source of the wheat D genome.It provide an important source of genetic diversity for wheat D genome.Here we present genome-wide methylation distribution and methylation density,and simultaneously examined the genetic and epigenetic structure in 115 Aegilops tauschii core accessions.We found that methylation is broad correlated with geography and climate of origin,suggesting that these DNA methylation variations may contribute to local adaptation.The methylation-climate association was strongly linked to CHG methylated sites and concentrated in transposable elements,while CG methylation associated with climate localized to genic regions.Notably,SUVH6 was found associated with genome-wide average m CHG levels and AGO61 associated with average m CG levels by GWAS analysis.This approach should be widely applicable for genetic linkage map construction,QTL mapping,genome-wide association analysis and genome-wide DNA methylation dissection in a variety of organisms and will improve the application in crop assisted breeding.