R. Appels

Characterization of polyploid wheat genomic diversity using a high-density 90 000 single nucleotide polymorphism array

High-density single nucleotide polymorphism (SNP) genotyping arrays are a powerful tool for studying genomic patterns of diversity, inferring ancestral relationships between individuals in populations and studying marker–trait associations in mapping experiments. We developed a genotyping array including about 90 000 gene-associated SNPs and used it to characterize genetic variation in allohexaploid and allotetraploid wheat populations. The array includes a significant fraction of common genome-wide distributed SNPs that are represented in populations of diverse geographical origin. We used density-based spatial clustering algorithms to enable high-throughput genotype calling in complex data sets obtained for polyploid wheat. We show that these model-free clustering algorithms provide accurate genotype calling in the presence of multiple clusters including clusters with low signal intensity resulting from significant sequence divergence at the target SNP site or gene deletions. Assays that detect low-intensity clusters can provide insight into the distribution of presence–absence variation (PAV) in wheat populations. A total of 46 977 SNPs from the wheat 90K array were genetically mapped using a combination of eight mapping populations. The developed array and cluster identification algorithms provide an opportunity to infer detailed haplotype structure in polyploid wheat and will serve as an invaluable resource for diversity studies and investigating the genetic basis of trait variation in wheat.

Aegilops tauschii draft genome sequence reveals a gene repertoire for wheat adaptation

About 8,000 years ago in the Fertile Crescent, a spontaneous hybridization of the wild diploid grass Aegilops tauschii (2n = 14; DD) with the cultivated tetraploid wheat Triticum turgidum (2n = 4x = 28; AABB) resulted in hexaploid wheat (T. aestivum; 2n = 6x = 42; AABBDD). Wheat has since become a primary staple crop worldwide as a result of its enhanced adaptability to a wide range of climates and improved grain quality for the production of baker’s flour. Here we describe sequencing the Ae. tauschii genome and obtaining a roughly 90-fold depth of short reads from libraries with various insert sizes, to gain a better understanding of this genetically complex plant. The assembled scaffolds represented 83.4% of the genome, of which 65.9% comprised transposable elements. We generated comprehensive RNA-Seq data and used it to identify 43,150 protein-coding genes, of which 30,697 (71.1%) were uniquely anchored to chromosomes with an integrated high-density genetic map. Whole-genome analysis revealed gene family expansion in Ae. tauschii of agronomically relevant gene families that were associated with disease resistance, abiotic stress tolerance and grain quality. This draft genome sequence provides insight into the environmental adaptation of bread wheat and can aid in defining the large and complicated genomes of wheat species.

A Physical Map of the 1-Gigabase Bread Wheat Chromosome 3B

As the staple food for 35% of the worlds population, wheat is one of the most important crop species. To date, sequence-based tools to accelerate wheat improvement are lacking. As part of the international effort to sequence the 17–billion–base-pair hexaploid bread wheat genome (2n = 6x = 42 chromosomes), we constructed a bacterial artificial chromosome (BAC)–based integrated physical map of the largest chromosome, 3B, that alone is 995 megabases. A chromosome-specific BAC library was used to assemble 82% of the chromosome into 1036 contigs that were anchored with 1443 molecular markers, providing a major resource for genetic and genomic studies. This physical map establishes a template for the remaining wheat chromosomes and demonstrates the feasibility of constructing physical maps in large, complex, polyploid genomes with a chromosome-based approach.

The wheat ribosomal DNA spacer region: Its structure and variation in populations and among species.

SummaryThe wheat rDNA clone pTA250 was examined in detail to provide a restriction enzyme map and the nucleotide sequence of two of the eleven, 130 bp repeating units found within the spacer region. The 130 bp units showed some sequence heterogeneity. The sequence difference between the two 130 bp units analysed (130.6 and 130.8) was at 7 positions and could be detected as a 4 °C shift in Tm when heterologous and homologous hybrids were compared. This corresponded to a 1.2% change in nucleotide sequence per ΔTm of 1 °C. The sensitivity of the Tm analysis using cloned sequences facilitated the analysis of small sequence variations in the spacer region of different Triticum aestivum cultivars and natural populations of T. turgidum ssp. dicoccoides (referred to as T. dicoccoides). In addition spacer length variation was assayed by restriction enzyme digestion and hybridization with spacer sequence probes.Extensive polymorphism was observed for the spacer region in various cultivars of T. aestivum, although within each cultivar the rDNA clusters were homogeneous and could be assigned to particular chromosomes. Within natural populations of T. dicoccoides polymorphism was also observed but, once again, within any one individual the rDNA clusters appeared to be homogeneous. The polymorphism, at the sequence level (assayed by Tm analysis), was not so great as to prevent the use of spacer sequence variation as a probe for evolutionary relationships. The length variation as assayed by restriction enzyme digestion did not appear to be as useful in this regard, since its range of variation was extensive even within populations of a species.

Megabase Level Sequencing Reveals Contrasted Organization and Evolution Patterns of the Wheat Gene and Transposable Element Spaces

This article describes the molecular analysis of large contiguous sequences produced from the bread wheat genome. It provides novel insights into the number, distribution, and density of genes along chromosome 3B and reveals an unexpectedly high amount of noncollinear genes compared to model grass genomes. To improve our understanding of the organization and evolution of the wheat (Triticum aestivum) genome, we sequenced and annotated 13-Mb contigs (18.2 Mb) originating from different regions of its largest chromosome, 3B (1 Gb), and produced a 2x chromosome survey by shotgun Illumina/Solexa sequencing. All regions carried genes irrespective of their chromosomal location. However, gene distribution was not random, with 75% of them clustered into small islands containing three genes on average. A twofold increase of gene density was observed toward the telomeres likely due to high tandem and interchromosomal duplication events. A total of 3222 transposable elements were identified, including 800 new families. Most of them are complete but showed a highly nested structure spread over distances as large as 200 kb. A succession of amplification waves involving different transposable element families led to contrasted sequence compositions between the proximal and distal regions. Finally, with an estimate of 50,000 genes per diploid genome, our data suggest that wheat may have a higher gene number than other cereals. Indeed, comparisons with rice (Oryza sativa) and Brachypodium revealed that a high number of additional noncollinear genes are interspersed within a highly conserved ancestral grass gene backbone, supporting the idea of an accelerated evolution in the Triticeae lineages.

When Genes Tell Different Stories: the Diploid Genera of Triticeae (Gramineae)

Gene trees are generally assumed to reflect the underlying phylogeny of the species, and they are, therefore, generally expected to be congruent with each other. Published gene trees from sequences of three nuclear genes-two 5S DNA arrays and the internal transcribed spacer (ITS) of the ribosomal RNA genes-are known to give somewhat different histories for diploid members of the Triticeae. A phylogeny based on cpDNA restriction sites gives yet another topology. Some discrepancies come from poorly supported nodes. Other differences, however, are statistically significant. The differences among the nuclear gene trees can be ascribed to particular taxa (notably Triticum monococcum and Aegilops tauschii), which have different histories for particular parts of their genomes. We have removed these two taxa, combined the three nuclear loci to produce a well-supported tree, and introduced the two species later as reticulations. The cpDNA tree has no groups in common with any of the nuclear gene trees, and is therefore not combined with them. Multiple reticulations must be introduced to reconcile the cpDNA and combined nuclear trees. Such dramatic differences among gene trees might be expected among populations in a species complex, but are surprising among intersterile genera.

Trends in genetic and genome analyses in wheat: a review

The size and structure of the wheat genome makes it one of the most complex crop species for genetic analysis. The development of molecular techniques for genetic analysis, in particular the use of molecular markers to monitor DNA sequence variation between varieties, landraces, and wild relatives of wheat and related grass species, has led to a dramatic expansion in our understanding of wheat genetics and the structure and behaviour of the wheat genome. This review provides an overview of these developments, examines some of the special issues that have arisen in applying molecular techniques to genetic studies in wheat, and looks at the applications of these technologies to wheat breeding and to improving our understanding of the genetic basis of traits such as disease resistance and processing quality. The review also attempts to foreshadow some of the key molecular issues and developments that may occur in wheat genetics and breeding over the next few years.

Construction of three linkage maps in bread wheat, Triticum aestivum

Genetic maps were compiled from the analysis of 160-180 doubled haploid lines derived from 3 crosses: Cranbrook × Halberd, CD87 × Katepwa, and Sunco × Tasman. The parental wheat lines covered a wide range of the germplasm used in Australian wheat breeding. The linkage maps were constructed with RFLP, AFLP, microsatellite markers, known genes, and proteins. The numbers of markers placed on each map were 902 for Cranbrook × Halberd, 505 for CD87 × Katepwa, and 355 for Sunco × Tasman. Most of the expected linkage groups could be determined, but 10-20% of markers could not be assigned to a specific linkage group. Homologous chromosomes could be aligned between the populations described here and linkage groups reported in the literature, based around the RFLP, protein, and microsatellite markers. For most chromosomes, colinearity of markers was found for the maps reported here and those recorded on published physical maps of wheat. AFLP markers proved to be effective in filling gaps in the maps. In addition, it was found that many AFLP markers defined specific genetic loci in wheat across all 3 populations. The quality of the maps and the density of markers differs for each population. Some chromosomes, particularly D genome chromosomes, are poorly covered. There was also evidence of segregation distortion in some regions, and the distribution of recombination events was uneven, with substantial numbers of doubled haploid lines in each population displaying one or more parental chromosomes. These features will affect the reliability of the maps in localising loci controlling some traits, particularly complex quantitative traits and traits of low heritability. The parents used to develop the mapping populations were selected based on their quality characteristics and the maps provide a basis for the analysis of the genetic control of components of processing quality. However, the parents also differ in resistance to several important diseases, in a range of physiological traits, and in tolerance to some abiotic stresses.

Controlled introgression to wheat of genes from rye chromosome arm 1RS by induction of allosyndesis : 2. Characterisation of recombinants.

SummaryChromosome pairing between rye chromosome arm 1RS, present in two wheat-rye translocation stocks, and its wheat homoeologues was induced by introducing the translocations into either a ph1bph1b or a nullisomic 5B background. This rye arm carries a gene conferring resistance to wheat stem rust, but lines carrying the translocation produce a poor quality dough unsuitable for breadmaking. Storage protein markers were utilised along with stem rust reaction to screen for allosyndetic recombinants. From a 1DL-1RS translocation, three lines involving wheat-rye recombination were recovered, along with thirteen lines derived from wheat-wheat homoeologous recombination. From a 1BL-1RS translocation, an additional three allosyndetic recombinants were recovered. Nullisomy for chromosome 5B was as efficacious as the ph1b mutant for induction of allosyndesis, and the former stock is easier to manipulate due to the presence of a 5BL-encoded endosperm protein. The novel wheat-rye chromosomes present in the recombinant lines may enable the rye disease resistance to be exploited without the associated dough quality defect.

Frequent duplication and deletion events in the 5S RNA genes and the associated spacer regions of theTriticeae

The 5 S DNA units from 15 grasses in theTriticeae were analysed at the DNA sequence level. Four units carried duplications near the 3′-end of the 5 S RNA gene with 3 of the duplications centred on the same base pairs as a duplication previously reported byGerlach & Dyer. The fourth duplication was located 3′ downstream from the gene, in the spacer region. Apparent deletions were very frequent when units of the different grasses were compared and it was clear that these deletions did not extend into a 75 bp spacer region upstream from the 5 S RNA gene. This 75 bp region also tended to be more conserved between the grasses as compared to the high level of sequence change in the rest of the spacer region. — Phenetic relationships were established between the grasses using the sequence data. The relationships were generally consistent with the data from other parameters and, in addition, showed that two Australian grasses were closely related to the other Northern hemisphere genera examined. The data concerning the Australian grasses is discussed in relation to the isolated nature of Australia.