High‐density genotyping of the A.E. Watkins Collection of hexaploid landraces identifies a large molecular diversity compared to elite bread wheat

The importance of wheat as a food crop makes it a major target for agricultural improvements. As one of the most widely grown cereal grains, together with maize and rice, wheat is the leading provider of calories in the global diet, constituting 29% of global cereal production in 2015. In the last few decades, however, yields have plateaued, suggesting that the green revolution, at least for wheat, might have run its course and that new sources of genetic variation are urgently required. The overall aim of our work was to identify novel variation that may then be used to enable the breeding process. As landraces are a potential source of such diversity, here we have characterized the A.E. Watkins Collection alongside a collection of elite accessions using two complementary high‐density and high‐throughput genotyping platforms. While our results show the importance of using the appropriate SNP collection to compare diverse accessions, they also show that the Watkins Collection contains a substantial amount of novel genetic diversity which has either not been captured in current breeding programmes or which has been lost through previous selection pressures. As a consequence of our analysis, we have identified a number of accessions which carry an array of novel alleles along with a number of interesting chromosome rearrangements which confirm the variable nature of the wheat genome.     

Retention of D genome chromosomes in pentaploid wheat crosses

The transfer of genes between Triticum aestivum (hexaploid bread wheat) and T. turgidum (tetraploid durum wheat) holds considerable potential for genetic improvement of both these closely related species. Five different T. aestivum/T. turgidum ssp. durum crosses were investigated using Diversity Arrays Technology (DArT) markers to determine the inheritance of parental A, B and D genome material in subsequent generations derived from these crosses. The proportions of A, B and D chromosomal segments inherited from the hexaploid parent were found to vary significantly among individual crosses. F(2) populations retained widely varying quantities of D genome material, ranging from 99% to none. The relative inheritance of bread wheat and durum alleles in the A and B genomes of derived lines also varied among the crosses. Within any one cross, progeny without D chromosomes in general had significantly more A and B genome durum alleles than lines retaining D chromosomes. The ability to select for and manipulate this non-random segregation in bread wheat/durum crosses will assist in efficient backcrossing of selected characters into the recurrent durum or hexaploid genotype of choice. This study illustrates the utility of DArT markers in the study of inter-specific crosses to commercial crop species.     

Genome‐wide association study for 13 agronomic traits reveals distribution of superior alleles in bread wheat from the Yellow and Huai Valley of China

Bread wheat is a leading cereal crop worldwide. Limited amount of superior allele loci restricted the progress of molecular improvement in wheat breeding. Here, we revealed new allelic variation distribution for 13 yield‐related traits in series of genome‐wide association studies (GWAS) using the wheat 90K genotyping assay, characterized in 163 bread wheat cultivars. Agronomic traits were investigated in 14 environments at three locations over 3 years. After filtering SNP data sets, GWAS using 20 689 high‐quality SNPs associated 1769 significant loci that explained, on average, ~20% of the phenotypic variation, both detected already reported loci and new promising genomic regions. Of these, repetitive and pleiotropic SNPs on chromosomes 6AS, 6AL, 6BS, 5BL and 7AS were significantly linked to thousand kernel weight, for example BS00021705_51 on 6BS and wsnp_Ex_c32624_41252144 on 6AS, with phenotypic variation explained (PVE) of ~24%, consistently identified in 12 and 13 of the 14 environments, respectively. Kernel length‐related SNPs were mainly identified on chromosomes 7BS, 6AS, 5AL and 5BL. Plant height‐related SNPs on chromosomes 4DS, 6DL, 2DS and 1BL were, respectively, identified in more than 11 environments, with averaged PVE of ~55%. Four SNPs were confirmed to be important genetic loci in two RIL populations. Based on repetivity and PVE, a total of 41 SNP loci possibly played the key role in modulating yield‐related traits of the cultivars surveyed. Distribution of superior alleles at the 41 SNP loci indicated that superior alleles were getting popular with time and modern cultivars had integrated many superior alleles, especially for peduncle length‐ and plant height‐related superior alleles. However, there were still 19 SNP loci showing less than percentages of 50% in modern cultivars, suggesting they should be paid more attention to improve yield‐related traits of cultivars in the Yellow and Huai wheat region. This study could provide useful information for dissection of yield‐related traits and valuable genetic loci for marker‐assisted selection in Chinese wheat breeding programme.     

CropSNPdb: a database of SNP array data for Brassica crops and hexaploid bread wheat

Advances in sequencing technology have led to a rapid rise in the genomic data available for plants, driving new insights into the evolution, domestication and improvement of crops. Single nucleotide polymorphisms (SNPs) are a major component of crop genomic diversity, and are invaluable as genetic markers in research and breeding programs. High‐throughput SNP arrays, or ‘SNP chips’, can generate reproducible sets of informative SNP markers and have been broadly adopted. Although there are many public repositories for sequencing data, which are routinely uploaded, there are no formal repositories for crop SNP array data. To make SNP array data more easily accessible, we have developed CropSNPdb ( http://snpdb.appliedbioinformatics.com.au ), a database for SNP array data produced by the Illumina Infinium? hexaploid bread wheat (Triticum aestivum) 90K and Brassica 60K arrays. We currently host SNPs from datasets covering 526 Brassica lines and 309 bread wheat lines, and provide search, download and upload utilities for users. CropSNPdb provides a useful repository for these data, which can be applied for a range of genomics and molecular crop‐breeding activities.     

Assessment of genetic diversity among Syrian durum (Triticum turgidum ssp. durum) and bread wheat (Triticum aestivum L.) using SSR markers

Genetic diversity among 49 wheat varieties (37 durum and 12 bread wheat) was assayed using 32 microsatellites representing 34 loci covering almost the whole wheat genome. The polymorphic information content (PIC) across the tested loci ranged from 0 to 0.88 with average values of 0.57 and 0.65 for durum and bread wheat respectively. B genome had the highest mean number of alleles (10.91) followed by A genome (8.3) whereas D genome had the lowest number (4.73). The correlation between PIC and allele number was significant in all genome groups accounting for 0.87, 074 and 0.84 for A, B and D genomes respectively, and over all genomes, the correlation was higher in tetraploid (0.8) than in hexaploid wheat varieties (0.5). The cluster analysis discriminated all varieties and clearly divided the two ploidy levels into two separate clusters that reflect the differences in genetic diversity within each cluster. This study demonstrates that microsatellites markers have unique advantages compared to other molecular and biochemical fingerprinting techniques in revealing the genetic diversity in Syrian wheat varieties that is crucial for wheat improvement.     

Sequencing and assembly of low copy and genic regions of isolated Triticum aestivum chromosome arm 7DS

The genome of bread wheat (Triticum aestivum) is predicted to be greater than 16 Gbp in size and consist predominantly of repetitive elements, making the sequencing and assembly of this genome a major challenge. We have reduced genome sequence complexity by isolating chromosome arm 7DS and applied second‐generation technology and appropriate algorithmic analysis to sequence and assemble low copy and genic regions of this chromosome arm. The assembly represents approximately 40% of the chromosome arm and all known 7DS genes. Comparison of the 7DS assembly with the sequenced genomes of rice (Oryza sativa) and Brachypodium distachyon identified large regions of conservation. The syntenic relationship between wheat, B. distachyon and O. sativa, along with available genetic mapping data, has been used to produce an annotated draft 7DS syntenic build, which is publicly available at http://www.wheatgenome.info . Our results suggest that the sequencing of isolated chromosome arms can provide valuable information of the gene content of wheat and is a step towards whole‐genome sequencing and variation discovery in this important crop.     

Assessment of genetic diversity among Syrian durum (<Emphasis Type="Italic">Triticum</Emphasis> ssp. <Emphasis Type="Italic">durum</Emphasis>) and bread wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.) using SSR markers

Genetic diversity among 49 wheat varieties (37 durum and 12 bread wheat) was assayed using 32 microsatellites representing 34 loci covering almost the whole wheat genome. The polymorphic information content (PIC) across the tested loci ranged from 0 to 0.88 with average values of 0.57 and 0.65 for durum and bread wheat respectively. B-genome had the highest mean number of alleles (10.91) followed by A genome (8.3) whereas D genome had the lowest number (4.73). The correlation between PIC and allele number was significant in all genome groups accounting for 0.87, 074 and 0.84 for A, B and D genomes respectively, and over all genomes, the correlation was higher in tetraploid (0.8) than in hexaploid wheat varieties (0.5). The cluster analysis discriminated all varieties and clearly divided the two ploidy levels into two separate clusters that reflect the differences in genetic diversity within each cluster. This study demonstrates that microsatellites markers have unique advantages compared to other molecular and biochemical fingerprinting techniques in revealing the genetic diversity in Syrian wheat varieties that is crucial for wheat improvement.     

The improved assembly of 7DL chromosome provides insight into the structure and evolution of bread wheat

Wheat is one of the most important staple crops worldwide and also an excellent model species for crop evolution and polyploidization studies. The breakthrough of sequencing the bread wheat genome and progenitor genomes lays the foundation to decipher the complexity of wheat origin and evolutionary process as well as the genetic consequences of polyploidization. In this study, we sequenced 3286 BACs from chromosome 7DL of bread wheat cv. Chinese Spring and integrated the unmapped contigs from IWGSC v1 and available PacBio sequences to close gaps present in the 7DL assembly. In total, 8043 out of 12 825 gaps, representing 3 491 264 bp, were closed. We then used the improved assembly of 7DL to perform comparative genomic analysis of bread wheat (Ta7DL) and its D donor, Aegilops tauschii (At7DL), to identify domestication signatures. Results showed a strong syntenic relationship between Ta7DL and At7DL, although some small rearrangements were detected at the distal regions. A total of 53 genes appear to be lost genes during wheat polyploidization, with 23% (12 genes) as RGA (disease resistance gene analogue). Furthermore, 86 positively selected genes (PSGs) were identified, considered to be domestication‐related candidates. Finally, overlapping of QTLs obtained from GWAS analysis and PSGs indicated that TraesCS7D02G321000 may be one of the domestication genes involved in grain morphology. This study provides comparative information on the sequence, structure and organization between bread wheat and Ae. tauschii from the perspective of the 7DL chromosome, which contribute to better understanding of the evolution of wheat, and supports wheat crop improvement.     

Gene editing of the wheat homologs of TONNEAU1‐recruiting motif encoding gene affects grain shape and weight in wheat

Grain size and weight are important components of a suite of yield‐related traits in crops. Here, we showed that the CRISPR‐Cas9 gene editing of TaGW7, a homolog of rice OsGW7 encoding a TONNEAU1‐recruiting motif (TRM) protein, affects grain shape and weight in allohexaploid wheat. By editing the TaGW7 homoeologs in the B and D genomes, we showed that mutations in either of the two or both genomes increased the grain width and weight but reduced the grain length. The effect sizes of mutations in the TaGW7 gene homoeologs coincided with the relative levels of their expression in the B and D genomes. The effects of gene editing on grain morphology and weight traits were dosage dependent with the double‐copy mutant showing larger effect than the respective single copy mutants. The TaGW7‐centered gene co‐expression network indicated that this gene is involved in the pathways regulating cell division and organ growth, also confirmed by the cellular co‐localization of TaGW7 with α‐ and β‐tubulin proteins, the building blocks of microtubule arrays. The analyses of exome capture data in tetraploid domesticated and wild emmer, and hexaploid wheat revealed the loss of diversity around TaGW7‐associated with domestication selection, suggesting that TaGW7 is likely to play an important role in the evolution of yield component traits in wheat. Our study showed how integrating CRISPR‐Cas9 system with cross‐species comparison can help to uncover the function of a gene fixed in wheat for allelic variants targeted by domestication selection and select targets for engineering new gene variants for crop improvement.     

Use of RFLPs to determine the chromosome composition of tetraploid triticale (A/B)(A/B)RR.

Tetraploid triticale, (A/B)(A/B)RR (2n = 28), is a botanical novelty, an amphiploid composed of a diploid rye and a 14 chromosome wheat genome made up of chromosomes of the A and B genomes of tetraploid wheat. Restriction fragment length polymorphism (RFLP) markers were used to elucidate the chromosome composition of the mixed wheat genome of 35 different tetraploid triticale lines. Of 128 possible A/B chromosome pair combinations, only 6 were found among these lines, with a prevalence of the 1A, 2A, 3B, 4B, 5B, 6B, and 7B karyotype. In most triticale lines stable wheat genomes made up of only homologous A or B genome chromosome pairs were identified, however, in some lines homoeologous chromosome pairs were found. In this paper we demonstrate that RFLPs can be used successfully as an alternative to C-banding for the identification of the chromosome composition of tetraploid triticale and discuss the possible selective advantage of specific chromosome composition.     

Dissecting the U,M, S and C genomes of wild relatives of bread wheat (Aegilops spp.) into chromosomes and exploring their synteny with wheat

Goat grasses (Aegilops spp.) contributed to the evolution of bread wheat and are important sources of genes and alleles for modern wheat improvement. However, their use in alien introgression breeding is hindered by poor knowledge of their genome structure and a lack of molecular tools. The analysis of large and complex genomes may be simplified by dissecting them into single chromosomes via flow cytometric sorting. In some species this is not possible due to similarities in relative DNA content among chromosomes within a karyotype. This work describes the distribution of GAA and ACG microsatellite repeats on chromosomes of the U, M, S and C genomes of Aegilops, and the use of microsatellite probes to label the chromosomes in suspension by fluorescence in situ hybridization (FISHIS). Bivariate flow cytometric analysis of chromosome DAPI fluorescence and fluorescence of FITC‐labelled microsatellites made it possible to discriminate all chromosomes and sort them with negligible contamination by other chromosomes. DNA of purified chromosomes was used as a template for polymerase chain reation (PCR) using Conserved Orthologous Set (COS) markers with known positions on wheat A, B and D genomes. Wheat–Aegilops macrosyntenic comparisons using COS markers revealed significant rearrangements in the U and C genomes, while the M and S genomes exhibited structure similar to wheat. Purified chromosome fractions provided an attractive resource to investigate the structure and evolution of the Aegilops genomes, and the COS markers assigned to Aegilops chromosomes will facilitate alien gene introgression into wheat.     

Analysis of gene-derived SNP marker polymorphism in US wheat (Triticum aestivum L.) cultivars

In this study, we developed 359 detection primers for single nucleotide polymorphisms (SNPs) previously discovered within intron sequences of wheat genes and used them to evaluate SNP polymorphism in common wheat (Triticum aestivum L.). These SNPs showed an average polymorphism information content (PIC) of 0.18 among 20 US elite wheat cultivars, representing seven market classes. This value increased to 0.23 when SNPs were pre-selected for polymorphisms among a diverse set of 13 hexaploid wheat accessions (excluding synthetic wheats) used in the wheat SNP discovery project (). PIC values for SNP markers in the D genome were approximately half of those for the A and B genomes. D genome SNPs also showed a larger PIC reduction relative to the other genomes (P < 0.05) when US cultivars were compared with the more diverse set of 13 wheat accessions. Within those accessions, D genome SNPs show a higher proportion of alleles with low minor allele frequencies (<0.125) than found in the other two genomes. These data suggest that the reduction of PIC values in the D genome was caused by differential loss of low frequency alleles during the population size bottleneck that accompanied the development of modern commercial cultivars. Additional SNP discovery efforts targeted to the D genome in elite wheat germplasm will likely be required to offset the lower diversity of this genome. With increasing SNP discovery projects and the development of high-throughput SNP assay technologies, it is anticipated that SNP markers will play an increasingly important role in wheat genetics and breeding applications. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Sequencing chromosome 5D of Aegilops tauschii and comparison with its allopolyploid descendant bread wheat (Triticum aestivum)

Flow cytometric sorting of individual chromosomes and chromosome‐based sequencing reduces the complexity of large, repetitive Triticeae genomes. We flow‐sorted chromosome 5D of Aegilops tauschii, the D genome donor of bread wheat and sequenced it by Roche 454 GS FLX platform to approximately 2.2x coverage. Repetitive sequences represent 81.09% of the survey sequences of this chromosome, and Class I retroelements are the prominent type, with a particular abundance of LTR/Gypsy superfamily. Nonrepetitive sequences were assembled to cover 17.76% of the total chromosome regions. Up to 6188 nonrepetitive gene loci were predicted to be encoded by the 5D chromosome. The numbers and chromosomal distribution patterns of tRNA genes suggest abundance in tRNA^Lys and tRNA^Met species, while the nonrepetitive assembly reveals tRNA^Ala species as the most abundant type. A comparative analysis of the genomic sequences of bread wheat and Aegilops chromosome 5D indicates conservation of gene content. Orthologous unique genes, matching Aegilops 5D sequences, numbered 3730 in barley, 5063 in Brachypodium, 4872 in sorghum and 4209 in rice. In this study, we provide a chromosome‐specific view into the structure and organization of the 5D chromosome of Ae. tauschii, the D genome ancestor of bread wheat. This study contributes to our understanding of the chromosome‐level evolution of the wheat genome and presents a valuable resource in wheat genomics due to the recent hybridization of Ae. tauschii genome with its tetraploid ancestor.     

Exploring the tertiary gene pool of bread wheat: sequence assembly and analysis of chromosome 5Mg of Aegilops geniculata

Next‐generation sequencing (NGS) provides a powerful tool for the discovery of important genes and alleles in crop plants and their wild relatives. Despite great advances in NGS technologies, whole‐genome shotgun sequencing is cost‐prohibitive for species with complex genomes. An attractive option is to reduce genome complexity to a single chromosome prior to sequencing. This work describes a strategy for studying the genomes of distant wild relatives of wheat by isolating single chromosomes from addition or substitution lines, followed by chromosome sorting using flow cytometry and sequencing of chromosomal DNA by NGS technology. We flow‐sorted chromosome 5M^g from a wheat/Aegilops geniculata disomic substitution line [DS5M^g (5D)] and sequenced it using an Illumina HiSeq 2000 system at approximately 50 × coverage. Paired‐end sequences were assembled and used for structural and functional annotation. A total of 4236 genes were annotated on 5M^g, in close agreement with the predicted number of genes on wheat chromosome 5D (4286). Single‐gene FISH indicated no major chromosomal rearrangements between chromosomes 5M^g and 5D. Comparing chromosome 5M^g with model grass genomes identified synteny blocks in Brachypodium distachyon, rice (Oryza sativa), sorghum (Sorghum bicolor) and barley (Hordeum vulgare). Chromosome 5M^g‐specific SNPs and cytogenetic probe‐based resources were developed and validated. Deletion bin‐mapped and ordered 5M^g SNP markers will be useful to track 5M‐specific introgressions and translocations. This study provides a detailed sequence‐based analysis of the composition of a chromosome from a distant wild relative of bread wheat, and opens up opportunities to develop genomic resources for wild germplasm to facilitate crop improvement.     

Phylogenetic relationships of Triticum and Aegilops and evidence for the origin of the A, B, and D genomes of common wheat (Triticum aestivum)

Common wheat (Triticum aestivum) has for decades been a textbook example of the evolution of a major crop species by allopolyploidization. Using a sophisticated extension of the PCR technique, we have successfully isolated two single-copy nuclear genes, DMC1 and EF-G, from each of the three genomes found in hexaploid wheat (BA(u)D) and from the two genomes of the tetraploid progenitor Triticum turgidum (BA(u)). By subjecting these sequences to phylogenetic analysis together with sequences from representatives of all the diploid Triticeae genera we are able for the first time to provide simultaneous and strongly supported evidence for the D genome being derived from Aegilops tauschii, the A(u) genome being derived from Triticum urartu, and the hitherto enigmatic B genome being derived from Aegilops speltoides. Previous problems of identifying the B genome donor may be associated with a higher diversification rate of the B genome compared to the A(u) genome in the polyploid wheats. The phylogenetic hypothesis further suggests that neither Triticum, Aegilops, nor Triticum plus Aegilops are monophyletic.     

Elucidating the genetic basis of biomass accumulation and radiation use efficiency in spring wheat and its role in yield potential

One of the major challenges for plant scientists is increasing wheat (Triticum aestivum) yield potential (YP). A significant bottleneck for increasing YP is achieving increased biomass through optimization of radiation use efficiency (RUE) along the crop cycle. Exotic material such as landraces and synthetic wheat has been incorporated into breeding programmes in an attempt to alleviate this; however, their contribution to YP is still unclear. To understand the genetic basis of biomass accumulation and RUE, we applied genome‐wide association study (GWAS) to a panel of 150 elite spring wheat genotypes including many landrace and synthetically derived lines. The panel was evaluated for 31 traits over 2 years under optimal growing conditions and genotyped using the 35K wheat breeders array. Marker‐trait association identified 94 SNPs significantly associated with yield, agronomic and phenology‐related traits along with RUE and final biomass (BM_PM) at various growth stages that explained 7%–17% of phenotypic variation. Common SNP markers were identified for grain yield, BM_PM and RUE on chromosomes 5A and 7A. Additionally, landrace and synthetic derivative lines showed higher thousand grain weight (TGW), BM_PM and RUE but lower grain number (GM2) and harvest index (HI). Our work demonstrates the use of exotic material as a valuable resource to increase YP. It also provides markers for use in marker‐assisted breeding to systematically increase BM_PM, RUE and TGW and avoid the TGW/GM2 and BM_PM/HI trade‐off. Thus, achieving greater genetic gains in elite germplasm while also highlighting genomic regions and candidate genes for further study.     

Genome‐wide selection footprints and deleterious variations in young Asian allotetraploid rapeseed

Brassica napus (AACC, 2n = 38) is an important oilseed crop grown worldwide. However, little is known about the population evolution of this species, the genomic difference between its major genetic groups, such as European and Asian rapeseed, and the impacts of historical large‐scale introgression events on this young tetraploid. In this study, we reported the de novo assembly of the genome sequences of an Asian rapeseed (B. napus), Ningyou 7, and its four progenitors and compared these genomes with other available genomic data from diverse European and Asian cultivars. Our results showed that Asian rapeseed originally derived from European rapeseed but subsequently significantly diverged, with rapid genome differentiation after hybridization and intensive local selective breeding. The first historical introgression of B. rapa dramatically broadened the allelic pool but decreased the deleterious variations of Asian rapeseed. The second historical introgression of the double‐low traits of European rapeseed (canola) has reshaped Asian rapeseed into two groups (double‐low and double‐high), accompanied by an increase in genetic load in the double‐low group. This study demonstrates distinctive genomic footprints and deleterious SNP (single nucleotide polymorphism) variants for local adaptation by recent intra‐ and interspecies introgression events and provides novel insights for understanding the rapid genome evolution of a young allopolyploid crop.     

Cell organisation,sulphur metabolism and ion transport-related genes are differentially expressed in Paracoccidioides brasiliensis mycelium and yeast cells

Background

Bread wheat (Triticum aestivum) is an important staple food. However, wheat gluten proteins cause celiac disease (CD) in 0.5 to 1% of the general population. Among these proteins, the α-gliadins contain several peptides that are associated to the disease.

Results

We obtained 230 distinct α-gliadin gene sequences from severaldiploid wheat species representing the ancestral A, B, and D genomes of the hexaploid bread wheat. The large majority of these sequences (87%) contained an internal stop codon. All α-gliadin sequences could be distinguished according to the genome of origin on the basis of sequence similarity, of the average length of the polyglutamine repeats, and of the differences in the presence of four peptides that have been identified as T cell stimulatory epitopes in CD patients through binding to HLA-DQ2/8. By sequence similarity, α-gliadins from the public database of hexaploid T. aestivum could be assigned directly to chromosome 6A, 6B, or 6D. T. monococcum (A genome) sequences, as well as those from chromosome 6A of bread wheat, almost invariably contained epitope glia-α9 and glia-α20, but never the intact epitopes glia-α and glia-α2. A number of sequences from T. speltoides, as well as a number of sequences fromchromosome 6B of bread wheat, did not contain any of the four T cell epitopes screened for. The sequences from T. tauschii (D genome), as well as those from chromosome 6D of bread wheat, were found to contain all of these T cell epitopes in variable combinations per gene. The differences in epitope composition resulted mainly from point mutations. These substitutions appeared to be genome specific.

Conclusion

Our analysis shows that α-gliadin sequences from the three genomes of bread wheat form distinct groups. The four known T cell stimulatory epitopes are distributed non-randomly across the sequences, indicating that the three genomes contribute differently to epitope content. A systematic analysis of all known epitopes in gliadins and glutenins will lead to better understanding of the differences in toxiCity among wheat varieties. On the basis of such insight, breeding strategies can be designed to generate less toxic varieties of wheat which may be tolerated by at least part of the CD patient population.