*

aState Key Laboratory of Plant Cell and Chromosome Engineering,Institute of Genetics and Developmental Biology,Chinese Academy of Sciences,Beijing 100101,China

bCollege of Life Sciences,University of Chinese Academy of Sciences,Beijing 100049,China

1.Introduction

Common wheat(Triticum aestivum L.),one of the important staple food crops in the world,feeds＞30%of the human population[1].The world annual production of wheat is＞620 million metric tons(http://www.fao.org/3/a-i4691e.pdf).China is the largest wheat producer and consumer.The Chinese annual wheat production is about 100 million tons[2].Wheat production needs to be constantly increased in order to satisfy the food demands of the increasing world.Accurate sequencing and assembly of wheat genomes are helpful for basic research and genetic improvement of wheat cultivars[3].

The different ecotypes of common wheat(such as winter wheat and spring wheat)adapt well to a wide range of climates.Wheat is an allohexaploid(AABBDD)that arose from two wide hybridization events.The first occurred 0.5–-3.0 million years ago between two diploid ancestral species carrying the A(T.urartu)and B(an unknown species)genomes and after chromosome doubling formed wild tetraploid wheat(Triticum turgidum ssp.dicoccoides,AABB).This species was domesticated to cultivated emmer(T.turgidum ssp.dicoccum,AABB)[4].The second hybridization tookplaceabout 9000 years ago between cultivated emmer and diploid goat grass(Aegilops tauschii,DD)to form allohexaploid common wheat[5].Compared with other major crops the common wheat genome is very large in size(～17 gigabases,Gb)with each subgenome being approximately 5.5 Gb[6]and complex in composition,of which＞80%is made up of repetitive sequences.Therefore,sequencing and assembly of the entire wheat genome was very challenging.During the past several years many effort was made in this area,and a series of draft and near completed genome/chromosome assemblies for common wheat and its diploid and tetraploid progenitors have been generated in succession[7–16].In this review,we mainly focus on contributions made by Chinese scientists in the area of wheat genome sequencing following a brief summary of international achievements.

2.International progress in wheat genome sequencing

In order to decode the mystery of the wheat genome and to expedite molecular breeding in wheat,a group of scientists and breeders initiated the International Wheat Genome Sequencing Consortium(IWGSC)in 2005.To overcome the difficulties caused by genome size and complexities,the 21 chromosomes of common wheat landrace Chinese Spring were separated by flow cytometric sorting.Bacterial artificial chromosome(BAC)libraries and physical maps were then constructed for each chromosome or chromosome arm.Chromosome sorting,DNA isolation and BAC library construction for each chromosome arm were performed in the laboratory of Prof.Jaroslav Dolezel at the Institute of Experimental Botany in the Czech Republic.Subsequent physical map construction and BAC sequencing were assigned to different laboratories of the International Wheat Genome Sequencing Consortium (IWGSC). Numerous projects by different groups were undertaken to produce reference sequences of single chromosome or chromosome arms.Chromosome 3B was the first chromosome to be sorted successfully due to its large size.A 3B physical map was generated using BAC clones originating from the purified 3B chromosome in 2008[17].BAC clones were selected by a minimal tiling path(MTP)approach and sequenced.The final pseudomolecule of 3B was 774 megabases(Mb)in length and carried 5326 protein-coding genes[13].Currently,all chromosomes/chromosome arms of Chinese Spring have been sorted and their physical maps have been constructed(http://www.wheatgenome.org/Projects/IWGSC-Bread-Wheat-Projects).

Sequences of many chromosomes,or parts thereof,are publicly available,including 1AS,1BS,3DS,5DS,7DS,1AL,1BL,4A,5A,6A,6B,and 7B[16,18–29].

In addition to the chromosome-based BAC-by-BAC sequencing strategy of IWGSC,Hall and colleagues in the UK applied a whole genome shotgun sequencing strategy with 454 pyro-sequencing technology to sequence Chinese Spring,and produced a five-fold coverage genome sequence of Chinese Spring in 2012.Based on the assemblies of 5.42 Gb,they predicted 94,000 to 96,000 genes,and assigned two-thirds of them to the three subgenomes(A,B,and D).The authors indicated that gene families were pronouncedly reduced in common wheat compared to the diploid progenitors,[7].Two years after the first wheat genome release,IWGSC published a chromosome-based draft sequence of Chinese Spring[1].Compared to the whole genome shotgun sequencing strategy,this approach differentiated the highly conserved gene copies in each chromosome.

The 21 chromosomes of Chinese Spring were isolated by flow cytometric sorting and sequenced by a chromosome-based shotgun sequencing strategy using Illumina technology to yield 10.2 Gb of genome sequence.Abundant gene losses and duplications were observed by intra-and inter-specific comparisons,indicating that the wheat genome was somewhat dynamic in evolution[1].In 2017,Clarketal.[11]published an improved genome sequence of Chinese Spring.They used precisely sized mate-pair libraries and an optimized algorithm to generate a new assembly representing＞78%of the genome,much higher than the scaffold proportion(～49%)produced previously by IWGSC.Genome-wide sequence rearrangements were revealed based on comparative analysis of the data.Zimin et al.[12]reported a more complete wheat genome assembly.The final sequences were generated by combining next generation(short Illumina reads)and third generation sequencing data(long Pacific Biosciences reads).＞15 Gb of final assembly representing＞90%of the Chinese Spring genome was created by merging two sets of sequences assembled using the MaSuRCA[30]and FALCON assemblers[31].This is the most complete wheat genome sequence published by far.Recently,IWGSC announced that they have completed a high quality sequence of Chinese Spring(IWGSC v1.0)and released the genomic data for public access (http://www.wheatgenome.org/News/Latest-news/RefSeq-v1.0-URGI).

In addition to sequencing Chinese Spring at the genome and single chromosome level,sequencing of wild emmer,the tetraploid ancestor of common wheat,was reported in July 2017[10].A software package Denovo MAGIC2(NRGene,NesZiona,Israel)was applied to perform thescaffold assembly from short Illumina sequencing reads.The software took advantage of improvements in throughput and read length of Illumina sequencing technology capable of completing challenging assemblies within days.About 10 Gb sequence of wild emmer genome was obtained by using whole genome shotgun sequencing of various insert-size libraries.The quality of the assembly was further validated by genetic data and three-dimensional chromosome conformation capture sequencing(Hi-C)data[32].Decoding the genome of the tetraploid ancestor will help in understanding the evolution of common wheat.

3.Wheat genome sequencing in China

Chinese scientists have made considerable contributions in wheat genome sequencing.They were first achieved to generate drafts of the A and D genomes in the diploid progenitors in 2013[8,9].These provided good reference information for generating the polyploid wheat genome assembly and analysis.

As described above,common wheat evolved from three diploid progenitors by two wide hybridization events.The diploid progenitors provide important foundations for understanding the evolution and domestication of hexaploid wheat.The progenitor species of the wheat A genome is T.urartu(wild einkorn)with an estimated genome size of 4.94 Gb.Ling et al.applied a whole genome shotgun sequencing strategy using an Illumina HiSequation(2000)sequencing platform,and successfully generated the draft genome of T.urartu with 4.66 Gb,accounting for 94%of the estimated genome size[9].The sequencing and assembly process was as follows:first,paired-end sequencing was performed to obtain genomic DNA sequence data from 57 DNA libraries with different insertion sizes.Then,low-quality,redundant and contaminated reads were removed to generate～91-fold coverage(448.49 Gb) high quality sequence data. Finally,the SOAPdenovo(version 1.05;http://soap.genomics.org.cn/)assembler was used to obtain 3.92 Gb of contigs with N50 size of 3.42 kilobases(kb)and the 4.66 Gb genome assembly with a scaffold N50 size of 63.69 kb.Assembly quality and coverage were validated using previously published BAC and expressed sequence tag(EST)sequences by PCR amplification.

About 67%of the assembly was annotated as repetitive elements,of which the most abundant were long terminal repeat retrotransposons (49.07%). In total, 34,879 protein-coding gene models were predicted by analyzing＞100 Mb of transcriptomes generated with the HiSequation and Roche 454 sequencing platform,together with publicly available ESTs from hexaploid wheat and related grass genomes[33–37].Average gene size was 3207 bp and exon number per gene was 4.7.Compared with 28,000 genes predicated for the A subgenome of hexaploid wheat[7]6800 more genes were identified in T.urartu.Although partly caused by different annotation methods the difference indicated extensive gene loss in the A subgenome of hexaploid wheat.Comparative analysis also revealed that there was a specific expansion of resistant genes in the T.urartu genome[9].Furthermore,the authors found that the large genome size of T.urartu compared to Brachypodium distachyon was caused by greatly increased intergenic spaces enriched with Gypsy and Copia retrotransposons,and firstly provided genome-scale evidence for the role of repeat expansion in genome size enlargement during the evolution of the tribe Triticeae[9].The T.urartu draft genome sequence will enable discovery of agronomic important genes and the development of genetic markers for molecular breeding.For example,TuGASR7 was shown to be a homologue of a grain length control gene in rice.Two haplotypes of TuGASR7(H1 and H2)were identified among 92 T.urartu accessions and H1 was significantly associated with long grain[9]and potentially higher yield if transferred to common wheat.

Genome sequencing of Ae.tauschii,the diploid progenitor of wheat D subgenome,was also reported by Chinese scientists[8].A whole genome shotgun sequencing strategy combined with paired-end Illumina sequencing was used to produce 557.55 Gb of raw data.Then low quality,adaptor-contaminated and PCR-duplicated reads were filtered to obtain a 378.86 Gb of high quality dataset.The Illumina reads were assembled by SOAPdenovo(version 1.05;http://soap.genomics.org.cn/)to yield 3.53 Gb of contigs with a N50 size of 4.5 kb.The assembled contigs were linked to a scaffold based on paired-end reads and an additional 18.4 Gb of 454 pyro-sequencing long reads.In total,4.23 Gb of scaffolds were achieved with a N50 size of 57.59 kb,representing 97%of the 4.36 Gb estimated genome size[8].The quality of the draft genome was evaluated by comparison with ESTs from two full-length cDNA libraries from leaf and root tissue of Ae.tauschii.About 91%of EST sequences were mapped to the scaffolds with＞90%coverage.

＞65.9%of the Ae.tauschii genome was annotated as repetitive DNA with the assemblies,[8].A large scale Ae.tauschii genome extension caused by a burst of retrotransposons was dated to have occurred about 3–-4 million years ago based on the insertion date of the assembled LTR retrotransposons.Jia et al.generated 117 Mb transcriptomes involving eight tissues to facilitate gene annotation[8].Using both evidence-based and de novo gene prediction methods,34,498 high confidence and 8652 low confidence protein-coding genes were identified.The latter have incomplete gene structure or limited expression data support.The average gene size was 1203 bp and exon number per gene was 4.9.About 1.72 Gb of scaffold sequences,comprising 30,697(71.1%)protein-coding genes,were aligned to chromosomes based on a genetic map constructed from an F2population of 490 individuals from a cross between Ae.tauschii accessions Y2280 and Al8/78.Genome-wide analysis revealed expansion of several agronomical relevant gene families,such as NBS-LRR and cytochrome P450 genes.

Researchers from China Agricultural University collaborating with several research groups in USA reported a high quality sequence of the short arm of Ae.tauschii chromosome 3D in 2017[15].To overcome the limitations of the whole genome shotgun sequencing strategy the authors used a BAC-by-BAC strategy to sequence 3176 BAC clones selected by MTP based on a previously reported Ae.tauschii physical map[38].These MTP clones were genetically anchored to the short arm of Ae.tauschii chromosome 3D,or co-assembled with the 3DS physical map of the common wheat.BAC DNA was used to construct 3 kb paired-end libraries and sequenced using the Roche GS FLX Titanium XL Chemistry protocol.Sequenced reads were assembled using the Roche 454 gs Assembler V2.6 package[39]and scaffolded by adding 3–5 kb mate-pair reads using the Consed package[40].The assembled sequences were merged according to MTP BAC order.A final assembly contained 689 scaffolds with a N50 of 766 kb and total length of 293 Mb.Two genetic maps[8,38]and a Radiation-Hybrid(RH)map[41]on3DS were used to build an At3DS pseudo molecule.In total,612 scaffolds(247 Mb)were anchored on to the pseudo molecule,covering 90%of the At3DS arm sequence.

＞81%of the At3DS pseudo molecule was annotated as transposable elements;this was higher than previously observated on the Ae. tauschii genome [8].Pericentromeric-centromeric region were localized from 170 to 247 Mb by plotting the density of the Cereba and Quinta repeat families.About 88%(1873)of the annotated high confidence protein-coding genes(2124)and 52%(51)of low confidence protein-coding genes(98)were expressed in Ae.tauschii.Average gene length was 3821 bp,which was longer than previous reports for the Ae.tauschii genome[8],and average exon number per gene was 4.3.At3DS was further compared with homologous segments in related grasses to reveal rapid evolution of Triticeae genomes.

4.New sequencing and assembling technologies promote progress in wheat genome sequencing

Past studies on wheat genome sequencing in China were mainly focused on whole genome or single chromosome arm of diploid ancestors.The most frequently used methods were second generation sequencing combined with whole genome shotgun or BAC-by-BAC strategies.The data produced have short read-lengths(＜300 bp)and biased genome coverage,resulting in fragmented and incomplete genome assemblies,especially in genomic regions with complex repeat structures.A new sequencing technology,single molecule real-time(SMRT)sequencing technology(Pacific Biosciences,USA),produces significantly longer sequence reads of up to 40–50 kb.This platform enables production of genome sequences with fewer gaps and longer contigs even for large and complicated genomes[42].Two additional next-generation mapping technologies,BioNano genome mapping and 10×Genomics linked reads,are being increasingly applied in genome sequencing to develop high quality assemblies[43–46].Moreover,a method described as Hi-C can be used to determine the three-dimensional architecture of chromosomes,providing insights into chromatin structure.Hi-C technology is able to validate genome assembly and scaffold order on chromosomes[32].

Combining these new technologies,our laboratory successfully generated high quality genome assemblies of T.urartu(in preparation for publication).We completed the genome sequencing using a BAC-by-BAC strategy combined with the SMRT sequencing technology and the new BioNano genome map and 10x Genomics linked reads mapping technologies.The assembly pipeline was briefly described as follows:Firstly,the Illumina clean reads in each BAC pool were separately assembled.Then,the sequence contigs of each BAC were connected using the best aligned PacBio reads.Thirdly,the BAC sequences were iteratively connected into FPC contigs based on the MTP physical map.Finally,the FPC contigs were merged into scaffolds by referring to BioNano consensus map,10x Genomics linked reads and mate-pair sequences.The assembly pipeline significantly increased the scaffold length and accuracy compared to the first version of the A genome sequence,where short reads sequencing data combined with whole genome shotgun sequencing were applied.Given the previously estimated genome size of 4.94 Gb,our new assembly accounts for 98.4%of the T.urartu genome.

As described above,the company NRGene(NesZiona,Isreal)developed the Denovo MAGIC2 assembler for large and complicated genomes such as wheat.It enables assembles of Illumina short reads into large scaffolds with N50 up to several Mb[10].Using this software,we completed the genome sequencing and assembly of Chinese bread wheat cultivar Kenong 9204.At the same time Prof.Jizeng Jia and colleagues at the Chinese Academy of Agricultural Sciences generated high quality Ae. tauschii genome assemblies,comprising of large scaffolds with a N50 size of 14.1 Mb[47].

5.Conclusions and perspectives

In general,the advent of new sequencing,mapping and assembly technologies will greatly facilitate the productions of high-quality genome sequences of Triticeae,and the genomes of various wheat species and cultivars will be sequenced and released for public access.These will strongly enhance systematic study of the genetics,comparative genomics and evolution of wheat,and will expedite isolation and characterization of genes controlling agronomical important traits,such as yield and resistance to biotic and abiotic stress.The complete genome sequence of common wheat and its progenitors will greatly assist molecular breeding of wheat and thereby contribute in meeting the challenges of food security and sustainable agriculture.

Acknowledgments

This work was supported by the Chinese Academy of Sciences(QYZDJ-SSW-SMC001)and the National Key Research and Development Program of China(2016YFD0101004).

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