Comparison of dot chromosome sequences from D. melanogaster and D. virilis reveals an enrichment of DNA transposon sequences in heterochromatic domains

Background

Chromosome four of Drosophila melanogaster, known as the dot chromosome, is largely heterochromatic, as shown by immunofluorescent staining with antibodies to heterochromatin protein 1 (HP1) and histone H3K9me. In contrast, the absence of HP1 and H3K9me from the dot chromosome in D. virilis suggests that this region is euchromatic. D. virilis diverged from D. melanogaster 40 to 60 million years ago.

Results

Here we describe finished sequencing and analysis of 11 fosmids hybridizing to the dot chromosome of D. virilis (372,650 base-pairs) and seven fosmids from major euchromatic chromosome arms (273,110 base-pairs). Most genes from the dot chromosome of D. melanogaster remain on the dot chromosome in D. virilis, but many inversions have occurred. The dot chromosomes of both species are similar to the major chromosome arms in gene density and coding density, but the dot chromosome genes of both species have larger introns. The D. virilis dot chromosome fosmids have a high repeat density (22.8%), similar to homologous regions of D. melanogaster (26.5%). There are, however, major differences in the representation of repetitive elements. Remnants of DNA transposons make up only 6.3% of the D. virilis dot chromosome fosmids, but 18.4% of the homologous regions from D. melanogaster; DINE-1 and 1360 elements are particularly enriched in D. melanogaster. Euchromatic domains on the major chromosomes in both species have very few DNA transposons (less than 0.4 %).

Conclusion

Combining these results with recent findings about RNAi, we suggest that specific repetitive elements, as well as density, play a role in determining higher-order chromatin packaging.     

Functional diversity of human protein kinase splice variants marks significant expansion of human kinome

Background

The low pH environment of the human stomach is lethal for most microorganisms; but not Escherichia coli, which can tolerate extreme acid stress. Acid resistance in E. coli is hierarchically controlled by numerous regulators among which are small noncoding RNAs (sncRNA).

Results

In this study, we individually deleted seventy-nine sncRNA genes from the E. coli K12-MG1655 chromosome, and established a single-sncRNA gene knockout library. By systematically screening the sncRNA mutant library, we show that the sncRNA GcvB is a novel regulator of acid resistance in E. coli. We demonstrate that GcvB enhances the ability of E. coli to survive low pH by upregulating the levels of the alternate sigma factor RpoS.

Conclusion

GcvB positively regulates acid resistance by affecting RpoS expression. These data advance our understanding of the sncRNA regulatory network involved in modulating acid resistance in E. coli.     

A novel T-DNA integration in rice involving two interchromosomal translocations

Key message

A male sterile transgenic rice plant TC-19 harboured a novel T-DNA integration in chromosome 8 with two interchromosomal translocations of 6.55 kb chromosome 3 and 29.8 kb chromosome 9 segments.

Abstract

We report a complex Agrobacterium T-DNA integration in rice (Oryza sativa) associated with two interchromosomal translocations. The T-DNA-tagged rice mutant TC-19, which harboured a single copy of the T-DNA, displayed male sterile phenotype in the homozygous condition. Analysis of the junctions between the T-DNA ends and the rice genome by genome walking showed that the right border is flanked by a chromosome 3 sequence and the left border is flanked by a chromosome 9 sequence. Upon further walking on chromosome 3, a chromosome 3/chromosome 8 fusion was detected. Genome walking from the opposite end of the chromosome 8 break point revealed a chromosome 8/chromosome 9 fusion. Our findings revealed that the T-DNA, together with a 6.55-kb region of chromosome 3 and a 29.8-kb region of chromosome 9, was translocated to chromosome 8. Southern blot analysis of the homozygous TC-19 mutant revealed that the native sequences of chromosome 3 and 9 were restored but the disruption of chromosome 8 in the first intron of the gene Os08g0152500 was not restored. The integration of the complex T-DNA in chromosome 8 caused male sterility.     

Genomic and small RNA sequencing of Miscanthus × giganteus shows the utility of sorghum as a reference genome sequence for Andropogoneae grasses

Background

Miscanthus × giganteus (Mxg) is a perennial grass that produces superior biomass yields in temperate environments. The essentially uncharacterized triploid genome (3n = 57, x = 19) of Mxg is likely critical for the rapid growth of this vegetatively propagated interspecific hybrid.

Results

A survey of the complex Mxg genome was conducted using 454 pyrosequencing of genomic DNA and Illumina sequencing-by-synthesis of small RNA. We found that the coding fraction of the Mxg genome has a high level of sequence identity to that of other grasses. Highly repetitive sequences representing the great majority of the Mxg genome were predicted using non-cognate assembly for de novo repeat detection. Twelve abundant families of repeat were observed, with those related to either transposons or centromeric repeats likely to comprise over 95% of the genome. Comparisons of abundant repeat sequences to a small RNA survey of three Mxg organs (leaf, rhizome, inflorescence) revealed that the majority of observed 24-nucleotide small RNAs are derived from these repetitive sequences. We show that high-copy-number repeats match more of the small RNA, even when the amount of the repeat sequence in the genome is accounted for.

Conclusions

We show that major repeats are present within the triploid Mxg genome and are actively producing small RNAs. We also confirm the hypothesized origins of Mxg, and suggest that while the repeat content of Mxg differs from sorghum, the sorghum genome is likely to be of utility in the assembly of a gene-space sequence of Mxg.     

Endogenous siRNAs and piRNAs derived from transposable elements and genes in the malaria vector mosquito Anopheles gambiae

Background

The siRNA and piRNA pathways have been shown in insects to be essential for regulation of gene expression and defence against exogenous and endogenous genetic elements (viruses and transposable elements). The vast majority of endogenous small RNAs produced by the siRNA and piRNA pathways originate from repetitive or transposable elements (TE). In D. melanogaster, TE-derived endogenous siRNAs and piRNAs are involved in genome surveillance and maintenance of genome integrity. In the medically relevant malaria mosquito Anopheles gambiae TEs constitute 12-16% of the genome size. Genetic variations induced by TE activities are known to shape the genome landscape and to alter the fitness in An. gambiae.

Results

Here, using bioinformatics approaches we analyzed the small RNA data sets from 6 libraries formally reported in a previous study and examined the expression of the mixed germline/somatic siRNAs and piRNAs produced in adult An. gambiae females. We characterized a large population of TE-derived endogenous siRNAs and piRNAs, which constitutes 56-60% of the total siRNA and piRNA reads in the analysed libraries. Moreover, we identified a number of protein coding genes producing gene-specific siRNAs and piRNAs that were generally expressed at much lower levels than the TE-associated small RNAs. Detailed sequence analysis revealed that An. gambiae piRNAs were produced by both “ping-pong” dependent (TE-associated piRNAs) and independent mechanisms (genic piRNAs). Similarly to D. melanogaster, more than 90% of the detected piRNAs were produced from TE-associated clusters in An. gambiae. We also found that biotic stress as blood feeding and infection with Plasmodium parasite, the etiological agent of malaria, modulated the expression levels of the endogenous siRNAs and piRNAs in An. gambiae.

Conclusions

We identified a large and diverse set of the endogenously derived siRNAs and piRNAs that share common and distinct aspects of small RNA expression across insect species, and inferred their impact on TE and gene activity in An. gambiae. The TE-specific small RNAs produced by both the siRNA and piRNA pathways represent an important aspect of genome stability and genetic variation, which might have a strong impact on the evolution of the genome and vector competence in the malaria mosquitoes.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1436-1) contains supplementary material, which is available to authorized users.     

DNA transposons and the role of recombination in mutation accumulation in Daphnia pulex

Background

We identify DNA transposons from the completed draft genome sequence of Daphnia pulex, a cyclically parthenogenetic, aquatic microcrustacean of the class Branchiopoda. In addition, we experimentally quantify the abundance of six DNA transposon families in mutation-accumulation lines in which sex is either promoted or prohibited in order to better understand the role of recombination in transposon proliferation.

Results

We identified 55 families belonging to 10 of the known superfamilies of DNA transposons in the genome of D. pulex. DNA transposons constitute approximately 0.7% of the genome. We characterized each family and, in many cases, identified elements capable of activity in the genome. Based on assays of six putatively active element families in mutation-accumulation lines, we compared DNA transposon abundance in lines where sex was either promoted or prohibited. We find the major difference in abundance in sexuals relative to asexuals in lab-reared lines is explained by independent assortment of heterozygotes in lineages where sex has occurred.

Conclusions

Our examination of the duality of sex as a mechanism for both the spread and elimination of DNA transposons in the genome reveals that independent assortment of chromosomes leads to significant copy loss in lineages undergoing sex. Although this advantage may offset the so-called 'two fold cost of sex' in the short-term, if insertions become homozygous at specific loci due to recombination, the advantage of sex may be decreased over long time periods. Given these results, we discuss the potential effects of sex on the dynamics of DNA transposons in natural populations of D. pulex.     

A BAC-based integrated linkage map of the silkworm Bombyx mori

Background

In 2004, draft sequences of the model lepidopteran Bombyx mori were reported using whole-genome shotgun sequencing. Because of relatively shallow genome coverage, the silkworm genome remains fragmented, hampering annotation and comparative genome studies. For a more complete genome analysis, we developed extended scaffolds combining physical maps with improved genetic maps.

Results

We mapped 1,755 single nucleotide polymorphism (SNP) markers from bacterial artificial chromosome (BAC) end sequences onto 28 linkage groups using a recombining male backcross population, yielding an average inter-SNP distance of 0.81 cM (about 270 kilobases). We constructed 6,221 contigs by fingerprinting clones from three BAC libraries digested with different restriction enzymes, and assigned a total of 724 single copy genes to them by BLAST (basic local alignment search tool) search of the BAC end sequences and high-density BAC filter hybridization using expressed sequence tags as probes. We assigned 964 additional expressed sequence tags to linkage groups by restriction fragment length polymorphism analysis of a nonrecombining female backcross population. Altogether, 361.1 megabases of BAC contigs and singletons were integrated with a map containing 1,688 independent genes. A test of synteny using Oxford grid analysis with more than 500 silkworm genes revealed six versus 20 silkworm linkage groups containing eight or more orthologs of Apis versus Tribolium, respectively.

Conclusion

The integrated map contains approximately 10% of predicted silkworm genes and has an estimated 76% genome coverage by BACs. This provides a new resource for improved assembly of whole-genome shotgun data, gene annotation and positional cloning, and will serve as a platform for comparative genomics and gene discovery in Lepidoptera and other insects.     

Profiling Sex-Specific piRNAs in Zebrafish

Piwi proteins and their partner small RNAs play an essential role in fertility, germ-line stem cell development, and the basic control and evolution of animal genomes. However, little knowledge exists regarding piRNA biogenesis. Utilizing microfluidic chip analysis, we present a quantitative profile of zebrafish piRNAs expressed differentially between testis and ovary. The sex-specific piRNAs are derived from separate loci of repeat elements in the genome. Ovarian piRNAs can be categorized into groups that reach up to 92 members, indicating a sex-specific arrangement of piRNA genes in the genome. Furthermore, precursor piRNAs preferentially form a hairpin structure at the 3′end, which seem to favor the generation of mature sex-specific piRNAs. In addition, the mature piRNAs from both the testis and the ovary are 2′-O-methylated at their 3′ ends.SMALL RNAs, ranging from 19 to 30 nucleotides (nt) in length, constitute a large family of regulatory molecules with diverse functions in invertebrates, vertebrates, plants, and fungi (Bartel 2004; Nakayashiki 2005). Two major classes of small RNAs are microRNAs (miRNAs) and small interfering RNAs (siRNAs). The functions of small RNAs have been conserved through evolution; they have been shown to inhibit gene expression at the levels of mRNA degradation, translational repression, chromatin modification, heterochromatin formation, and DNA elimination (Mochizuki et al. 2002; Bartel 2004; Kim et al. 2005; Brodersen and Voinnet 2006; Lee and Collins 2006; Vaucheret 2006).Over the past few years, focus on the genetics of small RNAs has helped clarify the mechanisms behind the regulation of these molecules. While hundreds of small RNAs have been identified from mammalian somatic tissues, relatively little is known about small RNAs in germ cells. A recent breakthrough has been the identification of small RNAs that associate with Piwi proteins (piRNAs) from Drosophila and mammalian gonads (Aravin et al. 2001, 2006; Girard et al. 2006; Grivna et al. 2006; Vagin et al. 2006; Watanabe et al. 2006). piRNAs and their interacting proteins Ziwi/Zili have also been identified in zebrafish (Houwing et al. 2007, 2008). Increasing evidence indicates that piRNAs play roles mainly in germ cell differentiation and genomic stability (Carthew 2006; Lau et al. 2006; Vagin et al. 2006; Brennecke et al. 2007; Chambeyron et al. 2008; Klattenhoff and Theurkauf 2008; Kuramochi-Miyagawa et al. 2008; Kim et al. 2009; Lim et al. 2009; Unhavaithaya et al. 2009). Moreover, although piRNAs are mostly expressed in germ line cells, recent studies showed piRNA expression in nongerm cells, for example, T-cell lines (Jurkat cells and MT4) (Azuma-Mukai et al. 2008; Yeung et al. 2009), indicating other functions such as in the immune system. piRNAs do not appear to be derived from double-stranded RNA precursors, and their biogenesis mechanisms, although unclear, may be distinct from those of siRNA and miRNA. Recently, two distinct piRNA production pathways were further proposed: the “ping-pong” model (Brennecke et al. 2007; Gunawardane et al. 2007) and the Ago3-independent piRNA pathway centered on Piwi in somatic cells (Li et al. 2009; Malone et al. 2009). However, the mechanistic pathways of piRNA activity and their biogenesis are still largely unknown.Teleost fishes comprise >24,000 species, accounting for more than half of extant vertebrate species, displaying remarkable variation in morphological and physiological adaptations (see review in Zhou et al. 2001). Recently, Houwing et al. (2007, 2008) reported findings on Ziwi/Zili and associated piRNAs, implicating roles in germ cell differentiation, meiosis, and transposon silencing in the germline of the zebrafish. However, some of the identified zebrafish piRNAs are nonrepetitive and nontransposon-related piRNAs, suggesting that piRNAs may have additional unknown roles. In this study, we show that for males and females, piRNAs are specifically derived from separate loci of the repeat elements, and that ovarian piRNAs are far more often associated in groups. Genomic analysis of piRNAs indicates a tendency to folding at the 3′ end of the piRNA precursor, which may favor cleavage of the piRNA precursor to generate mature sex-specific piRNAs. Furthermore, methylation modification occurs at the 2′-O-hydroxyl group on the ribose of the final 3′ nucleotide in both the testis and the ovary.     

Implantation Serine Proteinases heterodimerize and are critical in hatching and implantation

Background

Metamorphosis is a complex, highly conserved and strictly regulated development process that involves the programmed cell death of obsolete larval organs. Here we show a novel functional role for the aspartic proteinase cathepsin D during insect metamorphosis.

Results

Cathepsin D of the silkworm Bombyx mori (BmCatD) was ecdysone-induced, differentially and spatially expressed in the larval fat body of the final instar and in the larval gut of pupal stage, and its expression led to programmed cell death. Furthermore, BmCatD was highly induced in the fat body of baculovirus-infected B. mori larvae, suggesting that this gene is involved in the induction of metamorphosis of host insects infected with baculovirus. RNA interference (RNAi)-mediated BmCatD knock-down inhibited programmed cell death of the larval fat body, resulting in the arrest of larval-pupal transformation. BmCatD RNAi also inhibited the programmed cell death of larval gut during pupal stage.

Conclusion

Based on these results, we concluded that BmCatD is critically involved in the programmed cell death of the larval fat body and larval gut in silkworm metamorphosis.     

A major QTL associated with Fusarium oxysporum race 1 resistance identified in genetic populations derived from closely related watermelon lines using selective genotyping and genotyping-by-sequencing for SNP discovery

Key message

A major quantitative trait locus (QTL) for Fusarium oxysporum Fr. f. sp. niveum race 1 resistance was identified by employing a “selective genotyping” approach together with genotyping-by-sequencing technology to identify QTLs and single nucleotide polymorphisms associated with the resistance among closely related watermelon genotypes.

Abstract

Fusarium wilt is a major disease of watermelon caused by the soil-borne fungus Fusarium oxysporum Schlechtend.:Fr. f. sp. niveum (E.F. Sm.) W.C. Snyder & H.N. Hans (Fon). In this study, a genetic population of 168 F₃ families (24 plants in each family) exhibited continuous distribution for Fon race 1 response. Using a “selective genotyping” approach, DNA was isolated from 91 F₂ plants whose F₃ progeny exhibited the highest resistance (30 F₂ plants) versus highest susceptibility (32 F₂ plants), or moderate resistance to Fon race 1 (29 F₂ plants). Genotyping-by-sequencing (GBS) technology was used on these 91 selected F₂ samples to produce 266 single nucleotide polymorphism (SNP) markers, representing the 11 chromosomes of watermelon. A major quantitative trait locus (QTL) associated with resistance to Fon race 1 was identified with a peak logarithm of odds (LOD) of 33.31 and 1-LOD confidence interval from 2.3 to 8.4 cM on chromosome 1 of the watermelon genetic map. This QTL was designated “Fo-1.1” and is positioned in a genomic region where several putative pathogenesis-related or putative disease-resistant gene sequences were identified. Additional independent, but minor QTLs were identified on chromosome 1 (LOD 4.16), chromosome 3 (LOD 4.36), chromosome 4 (LOD 4.52), chromosome 9 (LOD 6.8), and chromosome 10 (LOD 5.03 and 4.26). Following the identification of a major QTL for resistance using the “selective genotyping” approach, all 168 plants of the F ₂ population were genotyped using the SNP nearest the peak LOD, confirming the association of this SNP marker with Fon race 1 resistance. The results in this study should be useful for further elucidating the mechanism of resistance to Fusarium wilt and in the development of molecular markers for use in breeding programs of watermelon.     

Identification of a robust molecular marker for the detection of the stem rust resistance gene Sr45 in common wheat

Key message

Fine mapping of the Ug99 effective stem rust resistance gene Sr45 introgressed into common wheat from the D -genome goatgrass Aegilops tauschii.

Abstract

Stem rust resistance gene Sr45, discovered in Aegilops tauschii, the progenitor of the D -genome of wheat, is effective against commercially important Puccinia graminis f. sp. tritici races prevalent in Australia, South Africa and the Ug99 race group. A synthetic hexaploid wheat (RL5406) generated by crossing Ae. tauschii accession RL5289 (carrying Sr45 and the leaf rust resistance gene Lr21) with a tetraploid experimental line ‘TetraCanthatch’ was previously used as the source in the transfer of these rust resistance genes to other hexaploid cultivars. Previous genetic studies on hexaploid wheats mapped Sr45 on the short arm of chromosome 1D with the following gene order: centromere–Sr45–Sr33–Lr21–telomere. To identify closely linked markers, we fine mapped the Sr45 region in a large mapping population generated by crossing CS1D5406 (disomic substitution line with chromosome 1D of RL5406 substituted for Chinese Spring 1D) with Chinese Spring. Closely linked markers based on 1DS-specific microsatellites, expressed sequence tags and AFLP were useful in the delineation of the Sr45 region. Sequences from an AFLP marker amplified a fragment that was linked with Sr45 at a distance of 0.39 cM. The fragment was located in a bacterial artificial chromosome clone of contig (ctg)2981 of the Ae. tauschii accession AL8/78 physical map. A PCR marker derived from clone MI221O11 of ctg2981 amplified 1DS-specific sequence that harboured an 18-bp indel polymorphism that specifically tagged the Sr45 carrying haplotype. This new Sr45 marker can be combined with a previously reported marker for Lr21, which will facilitate selecting Sr45 and Lr21 in breeding populations.