Whole-genome sequencing uncovers the structural and transcriptomic landscape of hexaploid wheat/Ambylopyrum muticum introgression lines

Wheat is a globally vital crop, but its limited genetic variation creates a challenge for breeders aiming to maintain or accelerate agricultural improvements over time. Introducing novel genes and alleles from wheat's wild relatives into the wheat breeding pool via introgression lines is an important component of overcoming this low variation but is constrained by poor genomic resolution and limited understanding of the genomic impact of introgression breeding programmes. By sequencing 17 hexaploid wheat/Ambylopyrum muticum introgression lines and the parent lines, we have precisely pinpointed the borders of introgressed segments, most of which occur within genes. We report a genome assembly and annotation of Am. muticum that has facilitated the identification of Am. muticum resistance genes commonly introgressed in lines resistant to stripe rust. Our analysis has identified an abundance of structural disruption and homoeologous pairing across the introgression lines, likely caused by the suppressed Ph1 locus. mRNAseq analysis of six of these introgression lines revealed that novel introgressed genes are rarely expressed and those that directly replace a wheat orthologue have a tendency towards downregulation, with no discernible compensation in the expression of homoeologous copies. This study explores the genomic impact of introgression breeding and provides a schematic that can be followed to characterize introgression lines and identify segments and candidate genes underlying the phenotype. This will facilitate more effective utilization of introgression pre-breeding material in wheat breeding programmes.     

A bacterial artificial chromosome contig spanning the major domestication locus Q in wheat and identification of a candidate gene

The Q locus played a major role in the domestication of wheat because it confers the free-threshing character and influences many other agronomically important traits. We constructed a physical contig spanning the Q locus using a Triticum monococcum BAC library. Three chromosome walking steps were performed by complete sequencing of BACs and identification of low-copy markers through similarity searches of database sequences. The BAC contig spans a physical distance of approximately 300 kb corresponding to a genetic distance of 0.9 cM. The physical map of T. monococcum had perfect colinearity with the genetic map of wheat chromosome arm 5AL. Recombination data in conjunction with analysis of fast neutron deletions confirmed that the contig spanned the Q locus. The Q gene was narrowed to a 100-kb segment, which contains an APETALA2 (AP2)-like gene that cosegregates with Q. AP2 is known to play a major role in controlling floral homeotic gene expression and thus is an excellent candidate for Q.     

Bat Guano Virome: Predominance of Dietary Viruses from Insects and Plants plus Novel Mammalian Viruses

Bats are hosts to a variety of viruses capable of zoonotic transmissions. Because of increased contact between bats, humans, and other animal species, the possibility exists for further cross-species transmissions and ensuing disease outbreaks. We describe here full and partial viral genomes identified using metagenomics in the guano of bats from California and Texas. A total of 34% and 58% of 390,000 sequence reads from bat guano in California and Texas, respectively, were related to eukaryotic viruses, and the largest proportion of those infect insects, reflecting the diet of these insectivorous bats, including members of the viral families Dicistroviridae, Iflaviridae, Tetraviridae, and Nodaviridae and the subfamily Densovirinae. The second largest proportion of virus-related sequences infects plants and fungi, likely reflecting the diet of ingested insects, including members of the viral families Luteoviridae, Secoviridae, Tymoviridae, and Partitiviridae and the genus Sobemovirus. Bat guano viruses related to those infecting mammals comprised the third largest group, including members of the viral families Parvoviridae, Circoviridae, Picornaviridae, Adenoviridae, Poxviridae, Astroviridae, and Coronaviridae. No close relative of known human viral pathogens was identified in these bat populations. Phylogenetic analysis was used to clarify the relationship to known viral taxa of novel sequences detected in bat guano samples, showing that some guano viral sequences fall outside existing taxonomic groups. This initial characterization of the bat guano virome, the first metagenomic analysis of viruses in wild mammals using second-generation sequencing, therefore showed the presence of previously unidentified viral species, genera, and possibly families. Viral metagenomics is a useful tool for genetically characterizing viruses present in animals with the known capability of direct or indirect viral zoonosis to humans.Bats belong to one of the most diverse, abundant, and widely distributed group of mammals. More than 1,100 bat species belong to the order of Chiroptera, representing approximately 20% of all mammalian species (54). Most bat species feed on insects and other arthropods, while others feed on fruit nectar, bird or mammal blood, and small vertebrates such as fish, frogs, mice, and birds (30). Of the 47 species of bats reported in the United States, most of them are insectivorous (http://www.batcon.org/).Bats are considered the natural reservoir of a large variety of zoonotic viruses causing serious human diseases such as lyssaviruses, henipaviruses, severe acute respiratory syndrome coronavirus, and Ebola virus (6, 38, 46, 59, 63, 65). Characteristics of bats, including their genetic diversity, broad geological distribution, gregarious habits, high population density, migratory habits, and long life span (30, 58), likely endow them with the ability to host diverse viruses, some of which are also able to infect humans and other mammals (41, 63).More than 80 virus species have been isolated or detected in bats using nucleic acid-based methods (6, 38, 59, 65). Viruses that have been recently discovered in bats include astroviruses, adeno-associated viruses (AAVs), adenoviruses, herpesviruses, and polyomavirus (8, 9, 13, 31, 32, 35, 37, 39, 40, 42, 61, 62, 68). For example, it was recently reported that a newly identified adenovirus isolated from bat guano was capable of infecting various vertebrate cell lines, including those of humans, monkeys, dogs, and pigs (35). With increasing human populations in previously wild areas, contact of bats with humans and with wild and domestic animals has increased, providing greater opportunities for cross-species transmissions of potentially pathogenic bat viruses. To better understand the range of viruses carried by bats, we undertook an initial characterization of the guano viromes of several common bat species in the United States.The development of massively parallel sequencing technology makes is possible to reveal uncultured viral assemblages within biological or environmental samples (11, 28). To date, this approach has been used to characterize viruses in equine feces (7), human blood (5), tissue (14), human feces (3, 4, 15, 45, 60, 67), and human respiratory secretions (64), which in turn has facilitated the discovery of many novel viruses (18, 20, 25, 33, 47, 50). In the present study, we analyzed the viruses present in guano from several bat species in California and Texas, using sequence-independent PCR amplification, pyrosequencing, and sequence similarity searches.     

Characterization of two genes expressed in the salivary glands of the Hessian fly, Mayetiola destructor (Say)

Two genes, SSGP-11A1 and SSGP-12A1, have been isolated that encodes proteins with a secretion signal peptide at theN-terminals from the Hessian fly (Mayetiola destructor (Say)). The SSGP-11A1 gene contains one small intron (89 bp) and encodes a putative protein with 79 amino acids. The first 18 amino acids constitute a putative secretion signal peptide. The SSGP-12A1 gene contains three small introns and encodes a putative protein with 234 amino acids. The first 19 amino acids constitute a putative secretion signal peptide. Northern blot analysis revealed that both of the genes are primarily expressed in the salivary glands of Hessian fly larvae, the feeding stage of the insect. These observations are consistent with the possibility that the proteins encoded by them are secreted into host plants during feeding. Even though both genes are exclusively expressed in Hessian fly larvae, the expression profiles between them were quite different in insects at different instars. The SSGP-11A1 gene was expressed in all instars of larvae while the SSGP-12A1 gene was almost exclusively expressed in the first instar larvae. The differential expression suggests that the proteins encoded by these two genes may perform different functions. In situ hybridization revealed that the SSGP-11A1 gene is located on the short arm of chromosome A1 while SSGP-12A1 gene is on the long arm of chromosome A2.     

Micro-colinearity between rice, <Emphasis Type="Italic">Brachypodium</Emphasis>, and <Emphasis Type="Italic">Triticum monococcum</Emphasis> at the wheat domestication locus <Emphasis Type="Italic">Q</Emphasis>

Brachypodium, a wild temperate grass with a small genome, was recently proposed as a new model organism for the large-genome grasses. In this study, we evaluated gene content and microcolinearity between diploid wheat (Triticum monococcum), Brachypodium sylvaticum, and rice at a local genomic region harboring the major wheat domestication gene Q. Gene density was much lower in T. monococcum (one per 41 kb) because of gene duplication and an abundance of transposable elements within intergenic regions as compared to B. sylvaticum (one per 14 kb) and rice (one per 10 kb). For the Q gene region, microcolinearity was more conserved between wheat and rice than between wheat and Brachypodium because B. sylvaticum contained two genes apparently not present within the orthologous regions of T. monococcum and rice. However, phylogenetic analysis of Q and leukotriene A-4 hydrolase-like gene orthologs, which were colinear among the three species, showed that Brachypodium is more closely related to wheat than rice, which agrees with previous studies. We conclude that Brachypodium will be a useful tool for gene discovery, comparative genomics, and the study of evolutionary relationships among the grasses but will not preclude the need to conduct large-scale genomics experiments in the Triticeae.     

The efficacy of Cot-based gene enrichment in wheat (Triticum aestivum L.).

We report the results of a study on the effectiveness of Cot filtration (CF) in the characterization of the gene space of bread wheat (Triticum aestivum L.), a large genome species (1C = 16,700 Mb) of tremendous agronomic importance. Using published Cot data as a guide, 2 genomic libraries for hexaploid wheat were constructed from the single-stranded DNA collected at Cot values > 1188 and 1639 M x s. Compared with sequences from a whole genome shotgun library from Aegilops tauschii (the D genome donor of bread wheat), the CF libraries exhibited 13.7-fold enrichment in genes, 5.8-fold enrichment in unknown low-copy sequences, and a 3-fold reduction in repetitive DNA. CF is twice as efficient as methylation filtration at enriching wheat genes. This research suggests that, with improvements, CF will be a highly useful tool in sequencing the gene space of wheat.