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July 7, 2019  |  

Cloning of the ?-secalin gene family in a wheat 1BL/1RS translocation line using BAC clone sequencing

Wheat 1BL/1RS translocation lines are planted around the world for their disease resistance and high yield. Most of them are poor in bread making, which is partially caused by ?-secalins that are encoded by the ?-secalin gene family, which is located on the short arm of rye chromosome 1R (1RS). However, information on the structure and evolution of the ?-secalin gene family is still limited.

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July 7, 2019  |  

Gene duplication confers enhanced expression of 27-kDa ?-zein for endosperm modification in quality protein maize.

The maizeopaque2(o2) mutant has a high nutritional value but it develops a chalky endosperm that limits its practical use. Genetic selection foro2modifiers can convert the normally chalky endosperm of the mutant into a hard, vitreous phenotype, yielding what is known as quality protein maize (QPM). Previous studies have shown that enhanced expression of 27-kDa ?-zein in QPM is essential for endosperm modification. Taking advantage of genome-wide association study analysis of a natural population, linkage mapping analysis of a recombinant inbred line population, and map-based cloning, we identified a quantitative trait locus (q?27) affecting expression of 27-kDa ?-zein.q?27was mapped to the same region as the majoro2 modifier(o2 modifier1) on chromosome 7 near the 27-kDa ?-zein locus.q?27resulted from a 15.26-kb duplication at the 27-kDa ?-zein locus, which increases the level of gene expression. This duplication occurred before maize domestication; however, the gene structure ofq?27appears to be unstable and the DNA rearrangement frequently occurs at this locus. Because enhanced expression of 27-kDa ?-zein is critical for endosperm modification in QPM,q?27is expected to be under artificial selection. This discovery provides a useful molecular marker that can be used to accelerate QPM breeding.

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July 7, 2019  |  

A pigeonpea gene confers resistance to Asian soybean rust in soybean.

Asian soybean rust (ASR), caused by the fungus Phakopsora pachyrhizi, is one of the most economically important crop diseases, but is only treatable with fungicides, which are becoming less effective owing to the emergence of fungicide resistance. There are no commercial soybean cultivars with durable resistance to P. pachyrhizi, and although soybean resistance loci have been mapped, no resistance genes have been cloned. We report the cloning of a P. pachyrhizi resistance gene CcRpp1 (Cajanus cajan Resistance against Phakopsora pachyrhizi 1) from pigeonpea (Cajanus cajan) and show that CcRpp1 confers full resistance to P. pachyrhizi in soybean. Our findings show that legume species related to soybean such as pigeonpea, cowpea, common bean and others could provide a valuable and diverse pool of resistance traits for crop improvement.

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July 7, 2019  |  

Suppressed recombination and unique candidate genes in the divergent haplotype encoding Fhb1, a major Fusarium head blight resistance locus in wheat.

Fine mapping and sequencing revealed 28 genes in the non-recombining haplotype containing Fhb1 . Of these, only a GDSL lipase gene shows a pathogen-dependent expression pattern. Fhb1 is a prominent Fusarium head blight resistance locus of wheat, which has been successfully introgressed in adapted breeding material, where it confers a significant increase in overall resistance to the causal pathogen Fusarium graminearum and the fungal virulence factor and mycotoxin deoxynivalenol. The Fhb1 region has been resolved for the susceptible wheat reference genotype Chinese Spring, yet the causal gene itself has not been identified in resistant cultivars. Here, we report the establishment of a 1 Mb contig embracing Fhb1 in the donor line CM-82036. Sequencing revealed that the region of Fhb1 deviates from the Chinese Spring reference in DNA size and gene content, which explains the repressed recombination at the locus in the performed fine mapping. Differences in genes expression between near-isogenic lines segregating for Fhb1 challenged with F. graminearum or treated with mock were investigated in a time-course experiment by RNA sequencing. Several candidate genes were identified, including a pathogen-responsive GDSL lipase absent in susceptible lines. The sequence of the Fhb1 region, the resulting list of candidate genes, and near-diagnostic KASP markers for Fhb1 constitute a valuable resource for breeding and further studies aiming to identify the gene(s) responsible for F. graminearum and deoxynivalenol resistance.

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July 7, 2019  |  

Genomic dark matter illuminated: Anopheles Y chromosomes.

Hall et al. have strategically used long-read sequencing technology to characterize the structure and highly repetitive content of the Y chromosome in Anopheles malaria mosquitoes. Their work confirms that this important but elusive heterochromatic sex chromosome is evolving extremely rapidly and harbors a remarkably small number of genes. Copyright © 2016 Elsevier Ltd. All rights reserved.

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July 7, 2019  |  

The Solanum demissum R8 late blight resistance gene is an Sw-5 homologue that has been deployed worldwide in late blight resistant varieties.

The potato late blight resistance gene R8 has been cloned. R8 is found in five late blight resistant varieties deployed in three different continents. R8 recognises Avr8 and is homologous to the NB-LRR protein Sw-5 from tomato. The broad spectrum late blight resistance gene R8 from Solanum demissum was cloned based on a previously published coarse map position on the lower arm of chromosome IX. Fine mapping in a recombinant population and bacterial artificial chromosome (BAC) library screening resulted in a BAC contig spanning 170 kb of the R8 haplotype. Sequencing revealed a cluster of at least ten R gene analogues (RGAs). The seven RGAs in the genetic window were subcloned for complementation analysis. Only one RGA provided late blight resistance and caused recognition of Avr8. From these results, it was concluded that the newly cloned resistance gene was indeed R8. R8 encodes a typical intracellular immune receptor with an N-terminal coiled coil, a central nucleotide binding site and 13 C-terminal leucine rich repeats. Phylogenetic analysis of a set of representative Solanaceae R proteins shows that R8 resides in a clearly distinct clade together with the Sw-5 tospovirus R protein from tomato. It was found that the R8 gene is present in late blight resistant potato varieties from Europe (Sarpo Mira), USA (Jacqueline Lee, Missaukee) and China (PB-06, S-60). Indeed, when tested under field conditions, R8 transgenic potato plants showed broad spectrum resistance to the current late blight population in the Netherlands, similar to Sarpo Mira.

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July 7, 2019  |  

Genome puzzle master (GPM): an integrated pipeline for building and editing pseudomolecules from fragmented sequences.

Next generation sequencing technologies have revolutionized our ability to rapidly and affordably generate vast quantities of sequence data. Once generated, raw sequences are assembled into contigs or scaffolds. However, these assemblies are mostly fragmented and inaccurate at the whole genome scale, largely due to the inability to integrate additional informative datasets (e.g. physical, optical and genetic maps). To address this problem, we developed a semi-automated software tool-Genome Puzzle Master (GPM)-that enables the integration of additional genomic signposts to edit and build ‘new-gen-assemblies’ that result in high-quality ‘annotation-ready’ pseudomolecules.With GPM, loaded datasets can be connected to each other via their logical relationships which accomplishes tasks to ‘group,’ ‘merge,’ ‘order and orient’ sequences in a draft assembly. Manual editing can also be performed with a user-friendly graphical interface. Final pseudomolecules reflect a user’s total data package and are available for long-term project management. GPM is a web-based pipeline and an important part of a Laboratory Information Management System (LIMS) which can be easily deployed on local servers for any genome research laboratory.The GPM (with LIMS) package is available at https://github.com/Jianwei-Zhang/LIMS CONTACTS: jzhang@mail.hzau.edu.cn or rwing@mail.arizona.eduSupplementary information: Supplementary data are available at Bioinformatics online. © The Author 2016. Published by Oxford University Press.

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July 7, 2019  |  

Strategies for sequence assembly of plant genomes

The field of plant genome assembly has greatly benefited from the development and widespread adoption of next-generation DNA sequencing platforms. Very high sequencing throughputs and low costs per nucleotide have considerably reduced the technical and budgetary constraints associated with early assembly projects done primarily with a traditional Sanger-based approach. Those improvements led to a sharp increase in the number of plant genomes being sequenced, including large and complex genomes of economically important crops. Although next-generation DNA sequencing has considerably improved our understanding of the overall structure and dynamics of many plant genomes, severe limitations still remain because next-generation DNA sequencing reads typically are shorter than Sanger reads. In addition, the software tools used to de novo assemble sequences are not necessarily designed to optimize the use of short reads. These cause challenges, common to many plant species with large genome sizes, high repeat contents, polyploidy and genome-wide duplications. This chapter provides an overview of historical and current methods used to sequence and assemble plant genomes, along with new solutions offered by the emergence of technologies such as single molecule sequencing and optical mapping to address the limitations of current sequence assemblies.

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July 7, 2019  |  

Third-generation sequencing and analysis of four complete pig liver esterase gene sequences in clones identified by screening BAC library.

Pig liver carboxylesterase (PLE) gene sequences in GenBank are incomplete, which has led to difficulties in studying the genetic structure and regulation mechanisms of gene expression of PLE family genes. The aim of this study was to obtain and analysis of complete gene sequences of PLE family by screening from a Rongchang pig BAC library and third-generation PacBio gene sequencing.After a number of existing incomplete PLE isoform gene sequences were analysed, primers were designed based on conserved regions in PLE exons, and the whole pig genome used as a template for Polymerase chain reaction (PCR) amplification. Specific primers were then selected based on the PCR amplification results. A three-step PCR screening method was used to identify PLE-positive clones by screening a Rongchang pig BAC library and PacBio third-generation sequencing was performed. BLAST comparisons and other bioinformatics methods were applied for sequence analysis.Five PLE-positive BAC clones, designated BAC-10, BAC-70, BAC-75, BAC-119 and BAC-206, were identified. Sequence analysis yielded the complete sequences of four PLE genes, PLE1, PLE-B9, PLE-C4, and PLE-G2. Complete PLE gene sequences were defined as those containing regulatory sequences, exons, and introns. It was found that, not only did the PLE exon sequences of the four genes show a high degree of homology, but also that the intron sequences were highly similar. Additionally, the regulatory region of the genes contained two 720bps reverse complement sequences that may have an important function in the regulation of PLE gene expression.This is the first report to confirm the complete sequences of four PLE genes. In addition, the study demonstrates that each PLE isoform is encoded by a single gene and that the various genes exhibit a high degree of sequence homology, suggesting that the PLE family evolved from a single ancestral gene. Obtaining the complete sequences of these PLE genes provides the necessary foundation for investigation of the genetic structure, function, and regulatory mechanisms of the PLE gene family.

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July 7, 2019  |  

Genome sequence and analysis of the Japanese morning glory Ipomoea nil.

Ipomoea is the largest genus in the family Convolvulaceae. Ipomoea nil (Japanese morning glory) has been utilized as a model plant to study the genetic basis of floricultural traits, with over 1,500 mutant lines. In the present study, we have utilized second- and third-generation-sequencing platforms, and have reported a draft genome of I. nil with a scaffold N50 of 2.88?Mb (contig N50 of 1.87?Mb), covering 98% of the 750?Mb genome. Scaffolds covering 91.42% of the assembly are anchored to 15 pseudo-chromosomes. The draft genome has enabled the identification and cataloguing of the Tpn1 family transposons, known as the major mutagen of I. nil, and analysing the dwarf gene, CONTRACTED, located on the genetic map published in 1956. Comparative genomics has suggested that a whole genome duplication in Convolvulaceae, distinct from the recent Solanaceae event, has occurred after the divergence of the two sister families.

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July 7, 2019  |  

BAC-pool sequencing and analysis confirms growth-associated QTLs in the Asian seabass genome.

The Asian seabass is an important marine food fish that has been cultured for several decades in Asia Pacific. However, the lack of a high quality reference genome has hampered efforts to improve its selective breeding. A 3D BAC pool set generated in this study was screened using 22 SSR markers located on linkage group 2 which contains a growth-related QTL region. Seventy-two clones corresponding to 22 FPC contigs were sequenced by Illumina MiSeq technology. We co-assembled the MiSeq-derived scaffolds from each FPC contig with error-corrected PacBio reads, resulting in 187 sequences covering 9.7?Mb. Eleven genes annotated within this region were found to be potentially associated with growth and their tissue-specific expression was investigated. Correlation analysis demonstrated that SNPs in ctsb, skp1 and ppp2ca can be potentially used as markers for selecting fast-growing fingerlings. Conserved syntenies between seabass LG2 and five other teleosts were identified. This study i) provided a 10?Mb targeted genome assembly; ii) demonstrated NGS of BAC pools as a potential approach for mining candidates underlying QTLs of this species; iii) detected eleven genes potentially responsible for growth in the QTL region; and iv) identified useful SNP markers for selective breeding programs of Asian seabass.

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July 7, 2019  |  

Interchromosomal core duplicons drive both evolutionary instability and disease susceptibility of the Chromosome 8p23.1 region.

Recurrent rearrangements of Chromosome 8p23.1 are associated with congenital heart defects and developmental delay. The complexity of this region has led to inconsistencies in the current reference assembly, confounding studies of genetic variation. Using comparative sequence-based approaches, we generated a high-quality 6.3-Mbp alternate reference assembly of an inverted Chromosome 8p23.1 haplotype. Comparison with nonhuman primates reveals a 746-kbp duplicative transposition and two separate inversion events that arose in the last million years of human evolution. The breakpoints associated with these rearrangements map to an ape-specific interchromosomal core duplicon that clusters at sites of evolutionary inversion (P = 7.8 × 10(-5)). Refinement of microdeletion breakpoints identifies a subgroup of patients that map to the same interchromosomal core involved in the evolutionary formation of the duplication blocks. Our results define a higher-order genomic instability element that has shaped the structure of specific chromosomes during primate evolution contributing to rearrangements associated with inversion and disease.© 2016 Mohajeri et al.; Published by Cold Spring Harbor Laboratory Press.

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July 7, 2019  |  

Chimeras link to tandem repeats and transposable elements in tetraploid hybrid fish

Abstract The formation of the allotetraploid hybrid lineage (4nAT) encompasses both distant hybridization and polyploidization processes. The allotetraploid offspring have two sets of sub-genomes inherited from both parental species and therefore it is important to explore its genetic structure. Herein, we construct a bacterial artificial chromosome library of allotetraploids, and then sequence and analyze the full-length sequences of 19 bacterial artificial chromosomes. Sixty-eight DNA chimeras are identified, which are divided into four models according to the distribution of the genomic DNA derived from the parents. Among the 68 genetic chimeras, 44 (64.71%) are linked to tandem repeats (TRs) and 23 (33.82%) are linked to transposable elements (TEs). The chimeras linked to TRs are related to slipped-strand mispairing and double-strand break repair while the chimeras linked to TEs are benefit from the intervention of recombinases. In addition, TRs and TEs are linked not only with the recombinations, but also with the insertions/deletions of DNA segments. We conclude that DNA chimeras accompanied by TRs and TEs coordinate a balance between the sub-genomes derived from the parents which reduces the genomic shock effects and favors the evolutionary and adaptive capacity of the allotetraploidization. It is the first report on the relationship between formation of the DNA chimeras and TRs and TEs in the polyploid animals.

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July 7, 2019  |  

New high copy tandem repeat in the content of the chicken W chromosome.

The content of repetitive DNA in avian genomes is considerably less than in other investigated vertebrates. The first descriptions of tandem repeats were based on the results of routine biochemical and molecular biological experiments. Both satellite DNA and interspersed repetitive elements were annotated using library-based approach and de novo repeat identification in assembled genome. The development of deep-sequencing methods provides datasets of high quality without preassembly allowing one to annotate repetitive elements from unassembled part of genomes. In this work, we search the chicken assembly and annotate high copy number tandem repeats from unassembled short raw reads. Tandem repeat (GGAAA)n has been identified and found to be the second after telomeric repeat (TTAGGG)n most abundant in the chicken genome. Furthermore, (GGAAA)n repeat forms expanded arrays on the both arms of the chicken W chromosome. Our results highlight the complexity of repetitive sequences and update data about organization of sex W chromosome in chicken.

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July 7, 2019  |  

Current advances in genome sequencing of common wheat and its ancestral species

Common wheat is an important and widely cultivated food crop throughout the world. Much progress has been made in regard to wheat genome sequencing in the last decade. Starting from the sequencing of single chromosomes/chromosome arms whole genome sequences of common wheat and its diploid and tetraploid ancestors have been decoded along with the development of sequencing and assembling technologies. In this review, we give a brief summary on international progress in wheat genome sequencing, and mainly focus on reviewing the effort and contributions made by Chinese scientists.

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