Wanlong Li

A 4-gigabase physical map unlocks the structure and evolution of the complex genome of Aegilops tauschii, the wheat D-genome progenitor

The current limitations in genome sequencing technology require the construction of physical maps for high-quality draft sequences of large plant genomes, such as that of Aegilops tauschii, the wheat D-genome progenitor. To construct a physical map of the Ae. tauschii genome, we fingerprinted 461,706 bacterial artificial chromosome clones, assembled contigs, designed a 10K Ae. tauschii Infinium SNP array, constructed a 7,185-marker genetic map, and anchored on the map contigs totaling 4.03 Gb. Using whole genome shotgun reads, we extended the SNP marker sequences and found 17,093 genes and gene fragments. We showed that collinearity of the Ae. tauschii genes with Brachypodium distachyon, rice, and sorghum decreased with phylogenetic distance and that structural genome evolution rates have been high across all investigated lineages in subfamily Pooideae, including that of Brachypodieae. We obtained additional information about the evolution of the seven Triticeae chromosomes from 12 ancestral chromosomes and uncovered a pattern of centromere inactivation accompanying nested chromosome insertions in grasses. We showed that the density of noncollinear genes along the Ae. tauschii chromosomes positively correlates with recombination rates, suggested a cause, and showed that new genes, exemplified by disease resistance genes, are preferentially located in high-recombination chromosome regions.

BAC-FISH in wheat identifies chromosome landmarks consisting of different types of transposable elements

Fluorescence in situ hybridization (FISH) has been widely used in the physical mapping of genes and chromosome landmarks in plants and animals. Bacterial artificial chromosomes (BACs) contain large inserts making them amenable for FISH mapping. We used BAC-FISH to study genome organization and evolution in hexaploid wheat and its relatives. We selected 56 restriction fragment length polymorphism (RFLP) locus-specific BAC clones from libraries of Aegilops tauschii (the D-genome donor of hexaploid wheat) and A-genome diploid Triticum monococcum. Different types of repetitive sequences were identified using BAC-FISH. Two BAC clones gave FISH patterns similar to the repetitive DNA family pSc119; one BAC clone gave a FISH pattern similar to the repetitive DNA family pAs1. In addition, we identified several novel classes of repetitive sequences: one BAC clone hybridized to the centromeric regions of wheat and other cereal species, except rice; one BAC clone hybridized to all subtelomeric chromosome regions in wheat, rye, barley and oat; one BAC clone contained a localized tandem repeat and hybridized to five D-genome chromosome pairs in wheat; and four BAC clones hybridized only to a proximal region in the long arm of chromosome 4A of hexaploid wheat. These repeats are valuable markers for defined chromosome regions and can also be used for chromosome identification. Sequencing results revealed that all these repeats are transposable elements (TEs), indicating the important role of TEs, especially retrotransposons, in genome evolution of wheat.

Comparative Analysis of Syntenic Genes in Grass Genomes Reveals Accelerated Rates of Gene Structure and Coding Sequence Evolution in Polyploid Wheat

Cycles of whole-genome duplication (WGD) and diploidization are hallmarks of eukaryotic genome evolution and speciation. Polyploid wheat (Triticum aestivum) has had a massive increase in genome size largely due to recent WGDs. How these processes may impact the dynamics of gene evolution was studied by comparing the patterns of gene structure changes, alternative splicing (AS), and codon substitution rates among wheat and model grass genomes. In orthologous gene sets, significantly more acquired and lost exonic sequences were detected in wheat than in model grasses. In wheat, 35% of these gene structure rearrangements resulted in frame-shift mutations and premature termination codons. An increased codon mutation rate in the wheat lineage compared with Brachypodium distachyon was found for 17% of orthologs. The discovery of premature termination codons in 38% of expressed genes was consistent with ongoing pseudogenization of the wheat genome. The rates of AS within the individual wheat subgenomes (21%–25%) were similar to diploid plants. However, we uncovered a high level of AS pattern divergence between the duplicated homeologous copies of genes. Our results are consistent with the accelerated accumulation of AS isoforms, nonsynonymous mutations, and gene structure rearrangements in the wheat lineage, likely due to genetic redundancy created by WGDs. Whereas these processes mostly contribute to the degeneration of a duplicated genome and its diploidization, they have the potential to facilitate the origin of new functional variations, which, upon selection in the evolutionary lineage, may play an important role in the origin of novel traits.

A diploid wheat TILLING resource for wheat functional genomics

BackgroundTriticum monococcum L., an A genome diploid einkorn wheat, was the first domesticated crop. As a diploid, it is attractive genetic model for the study of gene structure and function of wheat-specific traits. Diploid wheat is currently not amenable to reverse genetics approaches such as insertion mutagenesis and post-transcriptional gene silencing strategies. However, TILLING offers a powerful functional genetics approach for wheat gene analysis.ResultsWe developed a TILLING population of 1,532 M2 families using EMS as a mutagen. A total of 67 mutants were obtained for the four genes studied. Waxy gene mutation frequencies are known to be 1/17.6 - 34.4 kb DNA in polyploid wheat TILLING populations. The T. monococcum diploid wheat TILLING population had a mutation frequency of 1/90 kb for the same gene. Lignin biosynthesis pathway genes- COMT1, HCT2, and 4CL1 had mutation frequencies of 1/86 kb, 1/92 kb and 1/100 kb, respectively. The overall mutation frequency of the diploid wheat TILLING population was 1/92 kb.ConclusionThe mutation frequency of a diploid wheat TILLING population was found to be higher than that reported for other diploid grasses. The rate, however, is lower than tetraploid and hexaploid wheat TILLING populations because of the higher tolerance of polyploids to mutations. Unlike polyploid wheat, most mutants in diploid wheat have a phenotype amenable to forward and reverse genetic analysis and establish diploid wheat as an attractive model to study gene function in wheat. We estimate that a TILLING population of 5, 520 will be needed to get a non-sense mutation for every wheat gene of interest with 95% probability.

Recurrent Deletions of Puroindoline Genes at the Grain Hardness Locus in Four Independent Lineages of Polyploid Wheat

Polyploidy is known to induce numerous genetic and epigenetic changes but little is known about their physiological bases. In wheat, grain texture is mainly determined by the Hardness (Ha) locus consisting of genes Puroindoline a (Pina) and b (Pinb). These genes are conserved in diploid progenitors but were deleted from the A and B genomes of tetraploid Triticum turgidum (AB). We now report the recurrent deletions of Pina-Pinb in other lineages of polyploid wheat. We analyzed the Ha haplotype structure in 90 diploid and 300 polyploid accessions of Triticum and Aegilops spp. Pin genes were conserved in all diploid species and deletion haplotypes were detected in all polyploid Triticum and most of the polyploid Aegilops spp. Two Pina-Pinb deletion haplotypes were found in hexaploid wheat (Triticum aestivum; ABD). Pina and Pinb were eliminated from the G genome, but maintained in the A genome of tetraploid Triticum timopheevii (AG). Subsequently, Pina and Pinb were deleted from the A genome but retained in the Am genome of hexaploid Triticum zhukovskyi (AmAG). Comparison of deletion breakpoints demonstrated that the Pina-Pinb deletion occurred independently and recurrently in the four polyploid wheat species. The implications of Pina-Pinb deletions for polyploid-driven evolution of gene and genome and its possible physiological significance are discussed.

Expression of Lignin Biosynthetic Genes in Wheat during Development and upon Infection by Fungal Pathogens

As a major component of the cell wall, lignin plays an important role in plant development and defense response to pathogens, but negatively impacts biomass processability for biofuels. Silencing the target lignin genes for greater biomass processability should not significantly affect plant development and biomass yield but also must not compromise disease resistance. Here, we report experiments to identify a set of lignin genes that may be silenced without compromising disease resistance. We profiled the expression of 32 lignin biosynthetic candidate genes by qRT-PCR in 17 wheat tissues collected at three developmental stages. Twenty-one genes were expressed at a much higher level in stems compared to sheaths and leaf blades. Expression of seven these genes significantly correlated with lignin content. The co-expression patterns indicated that these 21 genes are under strong developmental regulation and may play a role in lignin biosynthesis. Profiling gene expression of same tissues challenged by two fungal pathogens, Fusarium graminearum and Puccina triticina indicated that expression of 17 genes was induced by F. graminearum. Only PAL1, a non-developmental-regulated gene, was induced by P. triticina. Thus, lignin biosynthetic pathway overlaps defense response to F. graminearum. Based on these criteria, 17 genes, F5H1, F5H2, 4CL2, CCR2, COMT1, and COMT2 in particular that do not overlap with disease resistance pathway, may be the targets for downregulation.

W3 Is a New Wax Locus That Is Essential for Biosynthesis of β-Diketone, Development of Glaucousness, and Reduction of Cuticle Permeability in Common Wheat.

The cuticle plays important roles in plant development, growth and defense against biotic and abiotic attacks. Crystallized epicuticular wax, the outermost layer of cuticle, is visible as white-bluish glaucousness. In crops like barley and wheat, glaucousness is trait of adaption to the dry and hot cultivation conditions, and hentriacontane-14,16-dione (β-diketone) and its hydroxy derivatives are the major and unique components of cuticular wax in the upper parts of adult plants. But their biosynthetic pathway and physiological role largely remain unknown. In the present research, we identified a novel wax mutant in wheat cultivar Bobwhite. The mutation is not allelic to the known wax production gene loci W1 and W2, and designated as W3 accordingly. Genetic analysis localized W3 on chromosome arm 2BS. The w3 mutation reduced 99% of β-diketones, which account for 63.3% of the total wax load of the wild-type. W3 is necessary for β-diketone synthesis, but has a different effect on β-diketone hydroxylation because the hydroxy-β-diketones to β-diketone ratio increased 11-fold in the w3 mutant. Loss of β-diketones caused failure to form glaucousness and significant increase of cuticle permeability in terms of water loss and chlorophyll efflux in the w3 mutant. Transcription of 23 cuticle genes from five functional groups was altered in the w3 mutant, 19 down-regulated and four up-regulated, suggesting a possibility that W3 encodes a transcription regulator coordinating expression of cuticle genes. Biosynthesis of β-diketones in wheat and their implications in glaucousness formation and drought and heat tolerance were discussed. Key Message W3 is essential for β-diketone biosynthesis but suppresses its hydroxylation. Loss-of-function mutation w3 significantly increased cuticle permeability in terms of water loss and chlorophyll efflux.

The chloroplast view of the evolution of polyploid wheat.

Polyploid wheats comprise four species: Triticum turgidum (AABB genomes) and T. aestivum (AABBDD) in the Emmer lineage, and T. timopheevii (AAGG) and T. zhukovskyi (AAGGA(m) A(m) ) in the Timopheevi lineage. Genetic relationships between chloroplast genomes were studied to trace the evolutionary history of the species. Twenty-five chloroplast genomes were sequenced, and 1127 plant accessions were genotyped, representing 13 Triticum and Aegilops species. The A. speltoides (SS genome) diverged before the divergence of T. urartu (AA), A. tauschii (DD) and the Aegilops species of the Sitopsis section. Aegilops speltoides forms a monophyletic clade with the polyploid Emmer and Timopheevi wheats, which originated within the last 0.7 and 0.4 Myr, respectively. The geographic distribution of chloroplast haplotypes of the wild tetraploid wheats and A. speltoides illustrates the possible geographic origin of the Emmer lineage in the southern Levant and the Timopheevi lineage in northern Iraq. Aegilops speltoides is the closest relative of the diploid donor of the chloroplast (cytoplasm), as well as the B and G genomes to Timopheevi and Emmer lineages. Chloroplast haplotypes were often shared by species or subspecies within major lineages and between the lineages, indicating the contribution of introgression to the evolution and domestication of polyploid wheats.

Evolution of New Disease Specificity at a Simple Resistance Locus in a Crop–Weed Complex: Reconstitution of the Lr21 Gene in Wheat

The wheat leaf-rust resistance gene Lr21 was first identified in an Iranian accession of goatgrass, Aegilops tauschii Coss., the D-genome donor of hexaploid bread wheat, and was introgressed into modern wheat cultivars by breeding. To elucidate the origin of the gene, we analyzed sequences of Lr21 and lr21 alleles from 24 wheat cultivars and 25 accessions of Ae. tauschii collected along the Caspian Sea in Iran and Azerbaijan. Three basic nonfunctional lr21 haplotypes, H1, H2, and H3, were identified. Lr21 was found to be a chimera of H1 and H2, which were found only in wheat. We attempted to reconstitute a functional Lr21 allele by crossing the cultivars Fielder (H1) and Wichita (H2). Rust inoculation of 5876 F2 progeny revealed a single resistant plant that proved to carry the H1H2 haplotype, a result attributed to intragenic recombination. These findings reflect how plants balance the penalty and the necessity of a resistance gene and suggest that plants can reuse “dead” alleles to generate new disease-resistance specificity, leading to a “death–recycle” model of plant-resistance gene evolution at simple loci. We suggest that selection pressure in crop–weed complexes contributes to this process.

Chromosome arm-specific BAC end sequences permit comparative analysis of homoeologous chromosomes and genomes of polyploid wheat

BackgroundBread wheat, one of the world’s staple food crops, has the largest, highly repetitive and polyploid genome among the cereal crops. The wheat genome holds the key to crop genetic improvement against challenges such as climate change, environmental degradation, and water scarcity. To unravel the complex wheat genome, the International Wheat Genome Sequencing Consortium (IWGSC) is pursuing a chromosome- and chromosome arm-based approach to physical mapping and sequencing. Here we report on the use of a BAC library made from flow-sorted telosomic chromosome 3A short arm (t3AS) for marker development and analysis of sequence composition and comparative evolution of homoeologous genomes of hexaploid wheat.ResultsThe end-sequencing of 9,984 random BACs from a chromosome arm 3AS-specific library (TaaCsp3AShA) generated 11,014,359 bp of high quality sequence from 17,591 BAC-ends with an average length of 626 bp. The sequence represents 3.2% of t3AS with an average DNA sequence read every 19 kb. Overall, 79% of the sequence consisted of repetitive elements, 1.38% as coding regions (estimated 2,850 genes) and another 19% of unknown origin. Comparative sequence analysis suggested that 70-77% of the genes present in both 3A and 3B were syntenic with model species. Among the transposable elements, gypsy/sabrina (12.4%) was the most abundant repeat and was significantly more frequent in 3A compared to homoeologous chromosome 3B. Twenty novel repetitive sequences were also identified using de novo repeat identification. BESs were screened to identify simple sequence repeats (SSR) and transposable element junctions. A total of 1,057 SSRs were identified with a density of one per 10.4 kb, and 7,928 junctions between transposable elements (TE) and other sequences were identified with a density of one per 1.39 kb. With the objective of enhancing the marker density of chromosome 3AS, oligonucleotide primers were successfully designed from 758 SSRs and 695 Insertion Site Based Polymorphisms (ISBPs). Of the 96 ISBP primer pairs tested, 28 (29%) were 3A-specific and compared to 17 (18%) for 96 SSRs.ConclusionThis work reports on the use of wheat chromosome arm 3AS-specific BAC library for the targeted generation of sequence data from a particular region of the huge genome of wheat. A large quantity of sequences were generated from the A genome of hexaploid wheat for comparative genome analysis with homoeologous B and D genomes and other model grass genomes. Hundreds of molecular markers were developed from the 3AS arm-specific sequences; these and other sequences will be useful in gene discovery and physical mapping.