Identification of SNPs in barley(Hordeum vulgare L.) by deep sequencing of six reduced representation libraries

*

aKey Laboratory ofCrop Germplasm Resources and Utilization(MOA),The NationalKey Facility for Crop Gene Resources and Genetic Improvement, Institute of Crop Science,Chinese Academy of Agricultural Sciences,Beijing 100081,China

bTibet Academy of Agriculturaland Animal Husbandry Sciences,Lhasa 850032,China

Identification of SNPs in barley(Hordeum vulgare L.) by deep sequencing of six reduced representation libraries

GanggangGuoa,DawaDondupa,b,LishaZhanga,ShaHua,XingmiaoYuana,JingZhanga,*

aKey Laboratory ofCrop Germplasm Resources and Utilization(MOA),The NationalKey Facility for Crop Gene Resources and Genetic Improvement, Institute of Crop Science,Chinese Academy of Agricultural Sciences,Beijing 100081,China

bTibet Academy of Agriculturaland Animal Husbandry Sciences,Lhasa 850032,China

A R T I C L E I N F O

Article history:

Received 21 February 2014

Received in revised form

11 June 2014

Accepted 3 July 2014

Available online 14 July 2014

Barley

SNP discovery

Reduced representation libraries

Allele-specific PCR

High-density genetic markers are required for genotyping and linkage mapping in identifying genes from crops with complex genomes,such as barley.As the most common variation,single nucleotide polymorphisms(SNPs)are suitable for accurate genotyping by using the next-generation sequencing(NGS)technology.Reduced representation libraries (RRLs)of five barley accessions and one mutant were sequenced using NGS technology for SNP discovery.Twenty million short reads were generated and the proportion of repetitive sequences was reduced by more than 56%.A total of 6061 SNPs were identified,and 451 were mapped to the draft sequence of the barley genome with pairing reads.Eleven SNPs were validated using length polymorphic allele-specific PCR markers.

?2014 Crop Science Society of China and Institute of Crop Science,CAAS.Production and hosting by Elsevier B.V.All rights reserved.

1.Introduction

Newly developed high-throughput SNP genotyping platforms have revolutionized genetic mapping and genome-wide association studies(GWAS)in plants[1,2]and animals[3].Biparental and association mapping populations are powerful genetic materials to study induced mutation and natural variation[4].SNPs are highly abundant genetic markers and are ideal for GWAS and genetic fine mapping[5].Genome re-sequencing-based SNP discovery relies on low-coverage sequencing of individual samples as well as the presence of a high-quality draft genome sequence[5].However,the cost of complete genome re-sequencing for SNP discovery is prohibitive,especially for species with large genomes.In order to meet this challenge genotyping methods based on next generation sequencing(NGS)have been developed and widely used,such as Complexity Reduction of Polymorphic Sequences(CRoPS) [6],Restriction site Associated DNA(RAD)[7],genotyping by sequencing(GBS)-narrow sense[8],and Multiplex Shotgun Genotyping(MSG)[9].It is particularly noteworthy that GBS has become a powerful tool for association studies and genomics-assisted breeding in a range of species including

those with complex genomes.As a popular GBS tool,the strategy of a restriction enzyme-based reduced representation library(RRL)is feasible and flexible for SNP identification because it reduces the complexity of the genome by orders of magnitude[10].RRLs were used for SNP discovery first in human genomics by Sanger sequencing[11].Later,as an efficient and cost-effective method,RRL was used in maize[12]and cattle[13] for SNP discovery by deep sequencing.

Barley,one of the first crops to be cultivated by humans,is the world's fourth-largest widely grown cereal.Its genome was sequenced in 2012[14].Among the sequenced genomes of major crops,high-density SNPs were developed from rice and maize by the re-sequencing method[2,15].However,SNP discovery in barley was limited to ESTs and unigene fragments in relevant germplasms or array-based transcriptome analysis [16–18].Development of genotyping by sequencing(GBS) technology was gradually optimized and adopted in barley for SNP identification and QTL mapping.Recently,a novel twoenzyme GBS protocol was developed and bi-parental populations were genotyped with GBS to develop SNPs in barley and wheat[19].To test new semiconductor sequencing platforms for GBS,Mascher et al.genotyped a recombinant inbred line (RIL)population of barley and concluded that GBS technology can easily be modified as an advanced sequencing technology and genomic analysis tool[20].A procedure for constructing GBS libraries by reducing genome complexity using restriction enzymes(REs)was reported.This procedure is simple,quick, highly reproducible with high specificity,and may reach important regions of the genome that are inaccessible by sequence capture approaches[8].In addition,a high-density consensus genetic map in barley was available and GWAS of morphological traits had been performed.A short awn gene, Breviaristatum-e(ari-e),was mapped to a smallgenetic intervalon chromosome 5H[21],and a master switch gene for anthocyanin production,ANTHOCYANINLESS 2(ANT2),encoding a basic helix–loop–helix protein(HvbHLH1)was also fine mapped[22]. The gene HvCEN,a homolog of Antirrhinum CENTRORADIALIS contributing to spring growth habit and environmental adaptation was identified in cultivated barley by the use of the 9K iSelect platform and GWAS[23].A highly specific in-solution hybridization-based whole exome capture platform was developed and it provides a powerful tool for re-sequencing the genomes of other accessions of barley and its relatives[24].In this study,we used the restriction enzyme-based RRL method and a parallel sequencing platform to discover de novo SNPs in six barley accessions.Some of the SNPs were converted into allele-specific PCR(AS-PCR)markers for marker validation. These converted markers have advantages of low cost per sample and ease of use,thus making them suitable for genetic diversity analysis of barley germplasm resources and markerassisted breeding.They can also be used in fine mapping of genes controlling important traits in barley.

2.Materials and methods

2.1.PlantgrowthandDNApreparation

Four barley germplasm accessions from China(ZDM01159, ZDM01467,ZDM00014,ZDM08324),one accession from Mexico (ZDM08233),and one mutant(93–597)with multi-node and stem branching,obtained byγ-irradiation ofthe accession ZDM08324, and selfing for fifteen generations,were used for RRL construction.Seeds of the six accessions were sterilized with 3%H2O2for 5 min,and washed three times for 5 min with purified water. Subsequently,they were germinated and grown in darkness at 18±2°C for 14 days.Etiolated seedlings were individually harvested and frozen in liquid nitrogen and then stored at -80°C for DNAextraction.DNA was extracted and purified with a DNeasy plant mini kit(Qiagen,Hilden,Germany).

2.2.RRLconstructionanddeepsequencing

Tenμg of DNA from each sample was digested with 100 units Mse I(New England Biolabs,Beverly,MA,USA)in a 200μL reaction system.In order to digest the sample completely,the reaction was carried out overnight at 37°C.The digested DNA was fractionated on a 3.0%agarose gel.Digestion products between 350 and 450 bp were recovered with a MinElute Gel Extraction Kit(Qiagen)according to the manufacturer's protocol.

Sequences were generated from the six barley RRLs on the Illumina GA IIDNA sequencing platform(Illumina,San Diego, CA,USA).Raw data were assigned to individual samples using the barcode sequence and trimmed to 40 bp at each end.For the sequencing of barcode ligation,ligation product amplification and sequencing were completed by the BIOMARKER Company(http://www.biomarker.com.cn/).

2.3.SNPdiscoveryandphylogeneticanalysis

The raw reads were firstly blasted against the Triticeae Repeat Sequence Database[25](TREP,http://wheat.pw.usda.gov/ITMI/ Repeats/).The matched sequences were filtered,and proportions of repetitive elements were evaluated.Non-repetitive reads were mapped against the whole genome shotgun assembly of barley cultivar Morex[14]with the CLC Genomics Workbench 6.02(http://www.clcbio.com/),and reads in pairs,or in broken pairs,and the average length of pairing reads were counted. Finally,the reads with＞3×coverage were used for polymorphism analysis and SNP discovery.All of the identified SNPs and their reference sequences(20 bp flanking sequence of mapped pairing reads)are listed in Supplemental Table S1. SNPs identified in at least four to six RRLs were used for phylogenetic analysis.The phylogenetic tree was constructed by the Dnapars program using PHYLIP software[26].

2.4.SNPvalidation

Twenty one SNPs distributed evenly on all7 barley chromosomes were randomly selected and converted to AS-PCR markers for SNP validation.Two pairs of primers were designed for identification and genotyping of each SNP as described previously[27]. The SNP is present at the 3′end of the allele-specific PCR primer to ensure specificity of amplification.Primer design was performed using WASP software[28](http://bioinfo.biotec.or.th/ WASP)with default parameters.The two primer pairs were multiplexed in a single-tube PCR assay to assess the allelic status at each SNP locus.Two AS-PCR products of different lengths were generated.The PCR products were electrophoresed on 1.5%agarose geland visualized under UV light.

Table 1–Summary of RRL sequence production and filtering.

3.Results

3.1.SNPdiscovery

The RRLs were sequenced from six representative barley accessions,and a total of 20 million raw reads were obtained by pair-end sequencing on an Illumina GA II.Raw data were assigned to individual samples using the barcode sequence and trimmed to 40-nucleotide high-quality sequences for further analysis.The number of raw sequence reads ranged from 2.38 million(for 93–597)to 4.18 million(ZDM00014),with an average of 3.3 million reads for the six barley genotypes. The proportions of repetitive sequences varied from 11.74% (ZDM08233)to 44.77%(ZDM08324),and the ratio of the genomic mapped reads varied from 15.26%(ZDM08233)to 36.93% (ZDM08324).The average length of pairing reads ranged from 344.23 bp(ZDM01467)to 411.52 bp(ZDM08324)among the six accessions.After removal of the repetitive and low quality reads,a total of 1.56 million high-quality reads(with over 3×depth)from 6 RRLs were used for polymorphism analysis and SNP discovery.Finally,6061 SNPs with a 4.82×average coverage were identified.The RRL library of ZDM08324 generatedthe most number of SNPs(4508)and ZDM08233 did the fewest (2045)(Table 1).Among which,451 SNPs can be mapped by paired-end reads to the draft sequence of the barley genome (Table S1).

Table 2–Repeat sequence content and composition in RRLs.

About 21%of reads contained repetitive elements,including 2.14%simple repeats,1.03%internal repeats,16.42%retroelements and 1.17%of DNA transposon.The content and frequency of repeats were examined and compared with previously reported data[29].The frequencies of all categories of repeats were reduced;in particular,the major class of repeat elements was reduced by more than 51%,and class IIrepetitive elements were reduced by about 5%,resulting in a total reduction in repetitive elements of 56%(Table 2).

The 1595 SNPs present in at least four libraries were used for phylogenetic analysis and construction of a phylogenetic tree(Fig.1).The six barley accessions clustered into two groups based on a genetic distance scale.One was a Chinese group,and the other was an introduced line.Although in the same cluster as the other three Chinese accessions,mutant 93–597 and its parent,ZDM08324,had the closest relationship as expected and clustered into a distinct sub-group.The other three Chinese accessions grouped together in a separate branch.

Fig.1–Diversity analysis of the 6 barley accessions.An extended majority rule consensus tree calculated using the Dnapars program from the Phylip package[18].Numbers on branches indicate bootstrap values.

3.2.SNPvalidation

Pair-end sequencing allowed us to perform further experimental validation.AS-PCR and agarose gelelectrophoresis were used for SNP marker detection.Since 7 of 21 SNPs did not satisfy the default parameter of primer design software,and 3 SNP markers did not generate specific products in multiplexed PCR amplification,only 11 AS-PCR markers(ICS_B1H_S0003, ICS_B2H_S0058,ICS_B3H_S0134,ICS_B3H_S0152,ICS_B3H_S0155, ICS_B4H_S0202,ICS_B5H_S0249,ICS_B5H_S0270,ICS_B7H_S0382, ICS_B7H_S0389,and ICS_B7H_S0400)were validated(Table 3). Different genotypes were clearly separated by length polymorphismofthe 11 SNP markers(Fig.2).These markers not only can be used to validate the SNP genotypes ofthe six sequenced barley accessions,butalso can be applied to identify genotypes ofbarley germplasm resources(Table 3).

4.Discussion

Reduced representation library(RRL)technology can reduce redundant parts of plant genomes for sequencing.In the present study,six RRLs were constructed and sequenced from six barley accessions for SNP discovery.Over 56%of the repetitive elements were reduced,indicating that RRLs were effective in the removalof repetitive sequences.Read mappingand SNP calling were performed against the draft genome sequence of cv.Morex.In comparison to the previous report of RAD sequencing in barley[30],similar numbers of SNPs were anchored to the barley genome.

Table 3–Sequences of allele-specific PCR primer pairs used for SNP validation.

Fig.2–Agarose gelbanding patterns for 11 AS-PCR markers.M:DL2000 DNA ladder.A–F:Accession IDs correspond to Table 3.

Because of the double impact of primer design and multiplex PCR amplification efficiency,the marker development success rate of length polymorphic AS-PCR was restricted,but,compared to other PCR-based SNP genotyping methods,such as TaqMan and KASP,AS-PCR is simple and cheap because it has no requirement for special detection instruments and fluorescent probes.Moreover,length polymorphic AS-PCR markers can be examined easily in the validation of SNPs. AS-PCR genotyping results also confirmed that depth sequencing ensures accurate of SNP calling.Furthermore, phylogenic analysis based on calling SNP markers revealed that ion mutant 93–597 and its parent(ZDM08324)had the closest relationship whereas the introduced showed the most distant genetic relationship.Our results proved that

the GBS method was a reliable and cost-effective approach for SNP identification and genotyping.

By combining deep sequencing and RRLs,we can focus on the non-repeated portion of the barley genome for SNP identification.Our results demonstrated that this approach can be used as a powerful genotyping platform for costeffective linkage analysis and GWAS instead of whole genome sequencing in cereals with large genomes.

5.Conclusion

High proportions of repetitive sequences increase the cost of SNP discovery by deep sequencing in barley and its relatives. The present study indicated that reduced representation libraries significantly reduce repetitive redundant DNA,thus reducing sequencing costs and enhancing the efficiency of SNP discovery.

Acknowledgments

This work was supported by the National Natural Science Foundation of China(31000711,31370032),China Agriculture Research System(CARS-05)and the Agricultural Science and Technology Innovation Program.

Supplementary material

Supplementary material related to this article can be found online at http://dx.doi.org/10.1016/j.cj.2014.06.008.Table S1-List of SNPs identified in the six barley RRLs.

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*Corresponding author.Tel.:+86 10 62189624.

E-mail address:zhangjing03@caas.cn(J.Zhang).

Peer review under responsibility of Crop Science Society of China and Institute of Crop Science,CAAS.

http://dx.doi.org/10.1016/j.cj.2014.06.008

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