Wheat functional genomics in the era of next generation sequencing: An update

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National Key Facility for Crop Gene Resources and Genetic Improvement,Institute of Crop Science,Chinese Academy of Agricultural Sciences,Beijing 100081,China

1.Introduction

Bread wheat or common wheat(Triticum aestivum L.)is one of the most important global foods.Common wheat originated by hybridization between cultivated tetraploid em mer(T.dicoccum,AABB)and diploid goat grass(Aegilops tauschii,DD)approximately 10,000 years ago[1].Wheat is one of the foundation species whose cultivation and domestication has been closely associated with the prosperity of agriculture and settled societies.Common wheat became one of the most widely grown crops due to its high yields and nutritional and processing qualities[2].By 2050 the world population will reach nine billion.The food needs of those people will require significantly increased wheat yield delivered by genetic improvement.This task is greatly assisted by advances in next-generation sequencing(NGS)technologies that will rapidly change wheat functional genomics studies and also make possible faster forward genetics studies.The wheat community has recently generated genome sequences not only for the wheat diploid donors of the A(T.urartu)[3]and D(Ae.tauschii)[4]genomes,but also the hexaploid wheat genome(T.aestivum)[5,6].More recently,updated genomic versions for Ae.tauschii[7]and Chinese Spring became available[8].Additional experimental approaches,resources,and computational tools are becoming available for gene identification that can be utilized in wheat breeding.

In this review we summarize recent progress on wheat genomics and functional genomics studies that was achieved with the assistance of the NGS technology that focused on whole genome sequencing of hexaploid wheat and its donor species,genomic polymorphism,cloning of genes of agronomic importance,and development of technical platforms.We envisage that the future of wheat functional genomics will be accelerated under the combined applications of new strategies for genetic mapping and new resources for discovering genetic variation.Identification of gene function will contribute significantly to wheat improvement.

2.Sequencing the large genomes of common wheat and its progenitors

A major advantage of NGS technologies is to make draft sequences of genomes,especially the larger ones,more affordable relative to that of traditional technologies.NGS technologies also provide more possibilities to investigate gene structure and expression.Heritable genome variation that underlies important agronomic traits can be identified in a quicker and systematic manner.Advances in NGS technology provide necessary tools to dissect large and complicated genomes such as hexaploid wheat.Even so,the large genome and polyploid nature of common wheat has been a huge challenge,especially the high percentage(80%–90%)of repetitive sequences[9,10].It is nearly impossible to distinguish sequences from the highly similar homoeologous sub-genomes that have diverged over 2.5–6.0 million years[11,12].Therefore,sequencing the genomes of the diploid progenitors became an alternative and complementary choice,resulting in genome sequences of Ae.tauschii and T.urartu,the D and A genome donors,respectively.These sequences provide the necessary genetic information to identify homoeologs in common wheat[3,4].

Due to the complexity of hexaploid wheat there were several early efforts to sequence the genome.An initial de novo assembly of sequence data was attempted in chromosome 7DS derived from flow-sorted chromosome arms,demonstrating that it was possible to assemble all known 7DS genes[13].The same approach confirmed the translocation between chromosome arms 7BS and 4AL in the Chinese Spring genome[14],that exemplified the types of genomic changes that occurred during early evolution and domestication[15].Such an approach was then applied to all wheat chromosome arms with the exception of 3B[6],which was isolated as an intact chromosome,resulting in the first draft genome assemblies for wheat chromosomes.The genomes of two additional cultivars,Opata M85 and W7984,were shotgun-sequenced by Illumina Solexa,albeit with only limited annotation performed on the assemblies[16].Among these efforts,the chromosome 3B sequence represented the highest quality sequence,which was based on a physical map[17].A better picture of wheat chromosome structure and functional partitioning was obtained by studying these sequences.Study of genomic diversity among wild and domesticated accessions can reveal genomic regions bearing the signature of selection under domestication[18].In 2017,a 10.1-gigabase assembly of the 14 chromosomes of wild em mer(T.turgidumssp.dicoccoides)was produced using the NRGene genome assembly algorithm and provided a detailed analysis on the gene content,genome architecture,and genetic diversity of this related AB genome donor to common wheat[19].

The complete sequencing of these wheat genomes with the assistance of NGS technology are milestones for wheat biology and provide long needed resources for wheat functional genomics.Despite this,in order to link phenotypic traits to functional genes,wheat researchers had to work on additional technical platforms and resources such as mutant libraries,full-length cDNA clones,and SNP microarrays.

3.NGS-based genotyping reveals the genetic diversity of wheat

Sequencing mRNA pools is one of the most efficient single nucleotide polymorphism (SNP)-discovery approaches because it is economic and relatively straight-forward[20,21].Transcript assemblies can be utilized to develop probes for targeted resequencing of coding or low-copy genomic regions[21–23].This so-called exome capture approach can be used to target particular classes of genes as well[24,25].Using whole exome capture(WEC)developed from an early genome assembly of Chinese Spring,an expanded polymorphism discovery was performed by re-sequencing 62 diverse wheat lines[22].These wheat lines included landraces and cultivars so as to capture the genetic diversity of the major global wheat growing regions,gaining a better understanding of the selection that shaped current genetic diversity in wheat.

Further evidence supported the role of gene presence absence variation as causes of variation in agronomic traits.To explore genetic diversity across 18 wheat cultivars,Monte-negro et al.[26]produced animproved Chinese Spring reference genome sequence.Apangenome of 140,500±102 genes was predicted,with a core set of 81,070±1631 genes and an average of128,656genes in each cultivar.In addition to variation in gene presence or absence,over 36 million inter-varietal single nucleotide polymorphisms(SNPs)were identified.A less costly approach to dissect the genetic diversity of wheat accessions is genotyping-by-sequencing(GBS),a restriction enzyme-based approach that reduces the sequence complexity by sequencing only the short regions close to the restriction sites hence representing a small fraction of the entire genome.This method does not rely on a fixed set of SNPs,such that it can detect population-specific variants.The unbiased nature of GBS makes it attractive for breeders[27],despite the difficulty in downstream bioinformatics compared with those based on array-based technologies.However,NGS-based technologies have provided a large amount of genetic information for wheat,and have already contributed to wheat breeding by providing information that was previously almost impossible to obtain in wheat.For example,high-resolution genome-wide association studies(GWAS)became a routine tool.

4.Development of wheat SNP microarrays

Despite continually decreasing NGS prices the wheat genome was still too large to work with,both in cost and in data processing.Thus,SNP-arrays became the first means for wheat researchers to gain genome-wide insights on specific wheat accessions.Microarrays with their numerous SNPs are powerful tools to reveal genomic diversity.The information can be utilized to infer ancestral relationships between individuals in populations and to study marker-traits associations in mapping experiments.In the past few years,a series of SNP-microarrays with various densities were produced.

The first version of a wheat SNP microarray was developed from nine wheat accessions and used to genotype nearly 3000 world-wide accessions to reveal multiple targets of selection for improvement of land races and cultivars of common wheat[21].The 9K iSelect SNP array was used to genotype and phenotypically characterized a Chinese wheat mini-core collection of 262 accessions.Totals of 2420 and 2396 SNPs were detected in the respective A-and B-genome chromosomes.The results showed that intense selection(domestication and breeding)had stronger effects on A-genome than on B-genome chromosomes.Based on the available breeding pedigrees many yield-related blocks were traced back to the well-known Strampelli introduction from Italy about one century ago.Modern breeding has dramatically increased diversity in gene coding regions.Tagged SNP markers are potentially useful for marker assisted selection of such haplotype blocks as a wheat breeding strategy[28].

The second version of a widely used SNP-array,the 90K array,was developed from 19 bread wheat accessions of diverse geographical origin[29].This array contains a large number of SNPs distributed across the common wheat genome.Currently,52,607 markers are mapped[30].A density-based spatial clustering algorithm was developed such that high-throughput geno-type calling in complex data sets can be achieved.The assays can also detect low-intensity clusters,which may represent the presence-absence variation(PAV)in wheat populations.

The most recent SNP array contains about 630,517 SNPs(660K array)developed by low coverage sequencing of 192 common wheat and related species accessions,including 60 worldwide modern wheat varieties,72 wheat landraces,30 wild emmer accessions,and 30 Ae.tauschii accessions[31].SNPs were assigned to the Chinese Spring genome assembly(https://urgi.versailles.inra.fr/download/iwgsc/IWGSC

WGASequences/).Although more probes mean higher cost,they provide more genetic information.Ahigh-density genetic map was constructed displaying good synteny with the 90K and 820K(unpublished)consensus maps.This array is unique by having added diversity from wheat donor accessions that are more polymorphic than wheat cultivars alone.

SNP arrays represent convenient platforms to genotype large numbers of wheat accessions and are ideal for breeding applications[20,32,33].Smaller but representative and informative SNPs can further reduce experimental costs,such as the Breeders 35K Axiom Array available at CerealsDB[34].Moreover,array-based assays can be converted into genome-specific KASP markers[35],or STARP markers that are even better in terms of cost and accuracy[36].Since these SNPs are derived from a collection of germ plasm,including elite wheat cultivars,land races,progenitors and ancestral species,they should have much wider application.Furthermore,a large proportion of the SNPs is genetically mapped and therefore can be used in both breeding and research.

5.Genome-wide association studies in wheat

Genome-wide association study(GWAS)is an alternative approach to identify genetic variation in natural populations.It identifies multiple genetic recombination events that have occurred during divergence under both wild and domesticated conditions[37].The molecular mechanisms of how crop landraces adapt to different agroclimatic conditions are valuable information that can be applied in crop improvement.In common wheat,sequence-based GWAS are still rare.Most work has involved other more affordable technologies such as diversity arrays technology(DArT).Candidate loci for water-soluble carbohydrate accumulation,an important drought-avoidance characteristic in wheat,were identified using a GWAS approach[38].Significantly associated loci were identified on chromosomes 1A,1B,1D,2D,and 4A.These markers can be used in marker-assisted selection of drought tolerant wheat.In a recent study,723 landraces from 10 Chinese agro-ecological zones were evaluated for 23 agronomic traits in six environments[39].The GWAS was conducted using 52,303 DArT markers and 149 significant markers were identified for 21 agronomic traits,some of which were co-localized with 29 QTL associated with 15 traits,including previously reported genes for heading date,flag leaf width,peduncle length,and thousand kernel weight.Another GWAS was for pre-harvest sprouting(PHS)traits among 717 Chinese wheat landraces using 9740 DArT-seq and 178,803 SNP markers;194 landraces displayed high levels of PHS resistance with mean germination indices of＜0.2,among which only nine were white-grained.This indicated that both natural and artificial selection had impacted on PHS response in Chinese landraces[40].

GBS is another cost-effective approach for GWAS[41].Multiplexed samples can be tagged with barcodes and genome-wide molecular marker discovery can be coupled with genotyping.A population study of 177 Ae.tauschii accessions using GBS identified 11,489 single nucleotide polymorphic(SNP)markers that were used for genetic diversity analysis[42].GWAS analyses on 114 non-redundant accessions with 5249 SNP markers revealed 17 SNPs associated with grain size.Candidate genes for cell division and differentiation were predicted from the associated SNP markers.GWAS has been used in Ae.tauschii to study additional traits of economic importance such as cadmium stress[43],P-deficiency[44]and other morphological traits[45].SNPs in the format of microarrays are now becoming more frequently used for GWAS in wheat.Using the wheat 90K microarray Sun et al.[46]identified new allelic variation in 13 yield-related traits in 163 bread wheat cultivars.A total of 1769 significant loci explaining an average of～20%or higher of the phenotypic variation were identified,including previously reported loci and new genomic regions[46].

GWAS with the assistance of NGS should reveal genetic variation linked to features within populations of germplasm and locate loci responsible for disease response or environmental adaptation[47,48].Further improvement in methods of sampling and accuracy in phenotyping should enable GWAS to have a much larger role in wheat functional genomics.

6.Functional annotation of wheat genes by Targeted Induced Local Lesions in Genomes(TILLING)

For most wheat genes,there are three similar but redundant copies.Conventional gene identification methods used for diploid species,such as rice and maize,become problematic and cumbersome for use in wheat.For functional analysis,each of the three homoeologs has to be individually evaluated and their combined effect on phenotypes should be considered.In this regard,TILLING has become a major approach for wheat gene functional annotation of genes in wheat[49–53].The first TILLING platform was developed in the diploid wheat T.monococcum,a cultivated A-genome diploid wheat species.T.monococcum is a model to study traits,genes,and alleles in bread wheat.That TILLING population contained 1532 M2families derived from ethyl methanesulfonate(EMS)treatment[54].The overall mutation frequency was detected as one in every92 kb by testingfour genes;namely,the WAXY gene involved in starch synthesis and three lignin-biosynthesis-pathway genes-COMT1,HCT2,and 4CL1[55,56].In theory,knowledge of gene function in diploid wheat should be transferrable to hexaploid wheat,but polyploidization may complicate the mode of function due to the multiple homoeologous copies.Thus,it is necessary to make TILLING populations in polyploid wheat.Several TILLING populations have been reported for tetraploid and hexaploid wheat[57–59].A population of 1920 EMS-derived individuals was generated in both hexaploid and allotetraploid wheat,among which 246 mutant alleles were detected for one or other of each of the homoeologous waxy genes[60].These mutants were claimed to represent more genetic diversity in polyploid wheat than had been found in the preceding 25 resources for durum and bread wheat was conducted by Uauy et al.[59],where1368tetraploid and 1536 hexaploid M2plants were produced.The mutation densities were high with a 1/38 kb mutation rate for hexaploid and a 1/51 kb rate for the tetraploid.These TILLING plants were shown to be useful in identifying gene function[52].For example,mutations in SBEIIa enzymes generated non-transgenic wheat plants that conferred high amylose contents and novel starch functionality[57].In another case,mutations were found in SBEIIa for both durum and bread wheat varieties with similar changes in amylose and resistant starch contents[59].With rapid accumulation of wheat genomics information,hopefully every wheat gene will soon have an available mutant for functional studies.

Nevertheless,conventional TILLING platforms are tedious and time consuming[61].Application of NGS technology allowed sequencing of exome-captured DNA from 2735 tetraploid Kronos and hexaploid Cadenza EMS-induced mutants[62].This resource also provides users with online access(http://www.wheat-tilling.com/)to conveniently identify knockout alleles for over 90%of wheat genes.Moreover,predesigned SNP-based primers[35]are available to validate the mutations,which can be combined to develop double or triple null mutants in order to overcome the problem of redundant homoeologs.Such a resource makes stocks of mutant lines permanent and easy to access and should facilitate characterization of wheat gene function.

More recently,genome-editing technologies provided additional tools to create DNA variation in polyploid wheat[63].The complete editing of all three homoeologs of target genes has been reported[64–66].Further optimization should establish an efficient genome editing system for wheat,with more versatile applications such as allele replacement[67]or targeted gene insertion[64].

7.Dynamic wheat transcriptomes

Deciphering the wheat genome sequence is the first step in understanding its physical structure and composition,providing genomic infrastructure for mapping actively transcribed regions,especially during plant development and adaptation to biotic and abiotic stresses.Transcriptome profiling is to identify expression patterns of all genes and their products in the wheat genome.Such information will facilitate in-depth analysis of gene function and regulation,as well as their interacting networks in complex biological processes.For a long time,cDNA microarrays have been the major tool for study of wheat transcriptomes[68–70].However,expression information from microarrays is limited because of the fixed number of probes on them,and that is largely dependent on the quality of genome annotation.For cDNA arrays,expression levels are representative,indirect measurement of gene expression levels by hybridization signals are short on precision in determining real transcript numbers in specific tissue or cell lines[71].

Sequencing RNA pools using NGS technologies(RNA-seq)allows a thorough survey of the entire transcriptional landscape,revealing genome-wide gene activity and alternative splicing in a quantitative manner[72–76].A recent study by Pfeifer et al.set a good example for effective RNA-seq analysis that may provide more reliable and useful transcript expression information[77].This cell type-specific transcriptome profiling of homoeologous genes identified distinct co-expression clusters in the developing wheat grain.These expression patterns may reflect the spatiotemporal progression of gene activities during wheat endosperm development[77].No global genome dominance,which was expected prior to this work,was found,but there was cell type-and stage-dependent genome dominance.The work provided unprecedented information about subgenome interaction and its effect on gene transcription in individual cell types in wheat grains[77].Such analyses reveal expression patterns of the entire wheat gene model set and the relationship between gene expression, function, and regulation.Our laboratory recently studied the transcriptome profiles at four stages of early wheat reproductive development,from spikelet initiation to floral organ differentiation,and the results provided a first picture of gene regulatory networks in inflorescence development in wheat and highlighted potential targets for wheat yield improvement[72].

8.NGS-supported map-based cloning

Map-based cloning, the conventional forward-genetics approach,is an efficient method for identifying genes of agronomic importance in cereal crops.The change in the wheat genomics landscape with resources now including complete genome sequences and high-quality gene models(IWGSC R EFSEQ v.1.0;[6,8]),transcriptomics databases[78,79],and high-density SNP arrays[29,80]makes it much easier to conduct map-based cloning in wheat.As a result,fine mapping of QTL has been greatly facilitated with wheat genomic resources.For instance,a grain weight QTL Qtgw-cb.5A was mapped on chromosome 5A[81].It was then physically located using newly developed wheat genome sequences and genemodels.Detailed characterization of the QTL provided direct genetic evidence that expansion of pericarp cells was a determinant of final grain size[81].

In contrast to hexaploid wheat with its complex genome diploid,wheat relatives are more suitable for map-based gene cloning and inheritance studies.For example,Sr35,a gene for resistance to the widely virulent stem rust pathogen(Puccinia graminis f.sp.tritici(Pgt))race Ug99,was cloned from T.monococcum(AA)and was found to be absent in T.urartu(the donor of A-genome)and in polyploid wheat[82].Sr35 showed strong resistance to race Ug99 in common wheat after being transferred from diploid wheat by distant hybridization[82].

Male Sterile 2(Ms2),another recently cloned gene,was also identified by map-based cloning[83,84].Using transgenic wheat and EMS-mediated mutagenesis, the authors identified aerminal-repeatetrotransposonsiniature(TRIM)in the promoter region of Ms2 that was responsible for activation of the gene,leading to its anther-specific expression and consequent male sterility.Ms2 is an“orphan”gene in that no homolog has been found outside the Triticeae family.The cloning of Ms2 provides substantial potential to assemble practical pipelines for recurrent selection in wheat breeding and possible hybrid seed production.Mutant screening and NGS sequencing played important roles in the process of cloning of this gene.

9.Concluding remarks

The application of NGS technology is rapidly expanding our knowledge of plant genomics.This is particularly true for wheat where functional genomics would never have been possible using earlier sequencing technologies.NGS enables the quest for higher quality common wheat reference genomes and resequencing of a large number of cultivated wheat genotypes and wild relative accessions in the foreseeable future.With the availability of these rich genetic resources and high quality genotyping platforms wheat functional genomics study is entering a new phase.A comprehensive survey of genetic diversity in wheat,including landraces and wild relatives,will deepen our understanding of the genetic basis underlying domestication and evolution of this vital crop.The use of the NGS technology and informatics combined with information on genetic variation will provide the thrust for wheat to catch up with other crops in studies on functional genomics.The time for genomics-assisted wheat breeding is finally arriving.

Acknowledgments

We apologize to authors whose work could be included in this review due to space limitations.This work was supported in partby the NationalKey R&D Program ofChina(2016YFD0101004,2016YFD0100300).

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