Occasional paternal inheritance of the germline-restricted chromosome in songbirds.

Songbirds have one special accessory chromosome, the so-called germline-restricted chromosome (GRC), which is only present in germline cells and absent from all somatic tissues. Earlier work on the zebra finch (Taeniopygia guttata castanotis) showed that the GRC is inherited only through the female line-like the mitochondria-and is eliminated from the sperm during spermatogenesis. Here, we show that the GRC has the potential to be paternally inherited. Confocal microscopy using GRC-specific fluorescent in situ hybridization probes indicated that a considerable fraction of sperm heads (1 to 19%) in zebra finch ejaculates still contained the GRC. In line with these cytogenetic data, sequencing of ejaculates revealed that individual males from two families differed strongly and consistently in the number of GRCs in their ejaculates. Examining a captive-bred male hybrid of the two zebra finch subspecies (T. g. guttata and T. g. castanotis) revealed that the mitochondria originated from a castanotis mother, whereas the GRC came from a guttata father. Moreover, analyzing GRC haplotypes across nine castanotis matrilines, estimated to have diverged for up to 250,000 y, showed surprisingly little variability among GRCs. This suggests that a single GRC haplotype has spread relatively recently across all examined matrilines. A few diagnostic GRC mutations that arose since this inferred spreading suggest that the GRC has continued to jump across matriline boundaries. Our findings raise the possibility that certain GRC haplotypes could selfishly spread through the population via occasional paternal transmission, thereby outcompeting other GRC haplotypes that were limited to strict maternal inheritance, even if this was partly detrimental to organismal fitness.

Satellitome comparison of two oedipodine grasshoppers highlights the contingent nature of satellite DNA evolution.

BACKGROUND: The full catalog of satellite DNA (satDNA) within a same genome constitutes the satellitome. The Library Hypothesis predicts that satDNA in relative species reflects that in their common ancestor, but the evolutionary mechanisms and pathways of satDNA evolution have never been analyzed for full satellitomes. We compare here the satellitomes of two Oedipodine grasshoppers (Locusta migratoria and Oedaleus decorus) which shared their most recent common ancestor about 22.8 Ma ago. RESULTS: We found that about one third of their satDNA families (near 60 in every species) showed sequence homology and were grouped into 12 orthologous superfamilies. The turnover rate of consensus sequences was extremely variable among the 20 orthologous family pairs analyzed in both species. The satDNAs shared by both species showed poor association with sequence signatures and motives frequently argued as functional, except for short inverted repeats allowing short dyad symmetries and non-B DNA conformations. Orthologous satDNAs frequently showed different FISH patterns at both intra- and interspecific levels. We defined indices of homogenization and degeneration and quantified the level of incomplete library sorting between species. CONCLUSIONS: Our analyses revealed that satDNA degenerates through point mutation and homogenizes through partial turnovers caused by massive tandem duplications (the so-called satDNA amplification). Remarkably, satDNA amplification increases homogenization, at intragenomic level, and diversification between species, thus constituting the basis for concerted evolution. We suggest a model of satDNA evolution by means of recursive cycles of amplification and degeneration, leading to mostly contingent evolutionary pathways where concerted evolution emerges promptly after lineages split.

Satellite DNA Is an Inseparable Fellow Traveler of B Chromosomes.

Next-Generation Sequencing (NGS) has revealed that B chromosomes in several species are enriched in repetitive DNA, mostly satellite DNA (satDNA). This raises the question of whether satDNA is important to B chromosomes for functional reasons or else its abundance on Bs is simply a consequence of properties of B chromosomes such as their dispensability and late replication. Here we review current knowledge in this respect and contextualize it within the frame of practical difficulties to perform this kind of research, the most important being the absence of good full genome sequencing for B-carrying species, which is an essential requisite to ascertain the intragenomic origin of B chromosomes. Our review analysis on 16 species revealed that 38% of them showed B-specific satDNAs whereas only one of them (6%) carried an inter-specifically originated B chromosome. This shows that B-specific satDNA families can eventually evolve in intraspecifically arisen B chromosomes. Finally, the possibility of satDNA accumulation on B chromosomes for functional reasons is exemplified by B chromosomes in rye, as they contain B-specific satDNAs which are transcribed and occupy chromosome locations where they might facilitate the kind of drive shown by this B chromosome during pollen grain mitosis.

Gene expression changes elicited by a parasitic B chromosome in the grasshopper Eyprepocnemis plorans are consistent with its phenotypic effects.

Parasitism evokes adaptive physiological changes in the host, many of which take place through gene expression changes. This response can be more or less local, depending on the organ or tissue affected by the parasite, or else systemic when the parasite affects the entire host body. The most extreme of the latter cases is intragenomic parasitism, where the parasite is present in all host nuclei as any other genomic element. Here, we show the molecular crosstalk between a parasitic chromosome (also named B chromosome) and the host genome, manifested through gene expression changes. The transcriptome analysis of 0B and 1B females of the grasshopper Eyprepocnemis plorans, validated by a microarray experiment performed on four B-lacking and five B-carrying females, revealed changes in gene expression for 188 unigenes being consistent in both experiments. Once discarded B-derived transcripts, there were 46 differentially expressed genes (30 up- and 16 downregulated) related with the adaptation of the host genome to the presence of the parasitic chromosome. Interestingly, the functions of these genes could explain some of the most important effects of B chromosomes, such as nucleotypic effects derived from the additional DNA they represent, chemical defense and detoxification, protein modification and response to stress, ovary function, and regulation of gene expression. Collectively, these changes uncover an intimate host-parasite interaction between A and B chromosomes during crucial steps of gene expression and protein function.

Interpopulation spread of a parasitic B chromosome is unlikely through males in the grasshopper Eyprepocnemis plorans.

The near-neutral model of B chromosome evolution predicts that population invasion is quite fast. To test this prediction, in 1994, we introduced males of the grasshopper Eyprepocnemis plorans from a B-carrying population into a B-lacking population and monitored the evolution of B-chromosome frequency up to 2013. We observed fluctuating very low B frequency across years but, remarkably, the B chromosome introduced (the B2 variant) was found up to 1996 only, whereas the B1 variant was present from 1996 onwards, presumably introduced by fishermen using E. plorans males as bait. Effective introgression of genetic material from the donor population was evidenced by the presence of a satellite DNA on autosome 9 (up to 1999) and the presence of one individual in 2006 showing an ISSR marker profile being highly similar to that found in the donor population. This indicated that the males introduced by us effectively mated with resident females, but donor genes rapidly decreased in frequency after this non-recurrent migration event. Taken together, our results indicated: (i) that the non-recurrent migration event had a slight, transient genetic effect on the recipient population, which was diluted in only a few generations; and (ii) that even with recurrent migration (forced by fishermen) the B chromosome failed to increase in frequency. Bearing in mind that B chromosomes in this species drive through females only, we hypothesize that B chromosomes most likely failed invasion in both migration events because the migrating sex shows no B-drive.

High-throughput analysis of satellite DNA in the grasshopper Pyrgomorpha conica reveals abundance of homologous and heterologous higher-order repeats.

Satellite DNA (satDNA) constitutes an important fraction of repetitive DNA in eukaryotic genomes, but it is barely known in most species. The high-throughput analysis of satDNA in the grasshopper Pyrgomorpha conica revealed 87 satDNA variants grouped into 76 different families, representing 9.4% of the genome. Fluorescent in situ hybridization (FISH) analysis of the 38 most abundant satDNA families revealed four different patterns of chromosome distribution. Homology search between the 76 satDNA families showed the existence of 15 superfamilies, each including two or more families, with the most abundant superfamily representing more than 80% of all satDNA found in this species. This also revealed the presence of two types of higher-order repeats (HORs), one showing internal homologous subrepeats, as conventional HORs, and an additional type showing non-homologous internal subrepeats, the latter arising by the combination of a given satDNA family with a non-annotated sequence, or with telomeric DNA. Interestingly, the heterologous subrepeats included in these HORs showed higher divergence within the HOR than outside it, suggesting that heterologous HORs show poor homogenization, in high contrast with conventional (homologous) HORs. Finally, heterologous HORs can show high differences in divergence between their constituent subrepeats, suggesting the possibility of regional homogenization.

Quantitative sequence characterization for repetitive DNA content in the supernumerary chromosome of the migratory locust.

Repetitive DNA is a major component in most eukaryotic genomes but is ignored in most genome sequencing projects. Here, we report the quantitative composition in repetitive DNA for a supernumerary (B) chromosome, in the migratory locust (Locusta migratoria), by Illumina sequencing of genomic DNA from B-carrying and B-lacking individuals and DNA obtained from a microdissected B chromosome, as well as the physical mapping of some elements. B chromosome DNA of 94.9% was repetitive, in high contrast with the 64.1% of standard (A) chromosomes. B chromosomes are enriched in satellite DNA (satDNA) (65.2% of B-DNA), with a single satellite (LmiSat02-176) comprising 55% of the B. Six satDNAs were visualized by FISH on the B chromosome, and the only A chromosome carrying all these satellites was autosome 9, pointing to this chromosome, along with autosome 8 (which shares histone genes with the B) as putative ancestors of the B chromosome. We found several transposable elements (TEs) showing nucleotidic variation specific to B-carrying individuals, which was also present in B-carrying transcriptomes. Remarkably, an interstitial region of the B chromosome included a 17 kb chimera composed of 29 different TEs, suggesting reiterative TE insertion in this B chromosome region.

Post-meiotic B chromosome expulsion, during spermiogenesis, in two grasshopper species.

Most supernumerary (B) chromosomes are parasitic elements carrying out an evolutionary arms race with the standard (A) chromosomes. A variety of weapons for attack and defense have evolved in both contending elements, the most conspicuous being B chromosome drive and A chromosome drive suppression. Here, we show for the first time that most microspermatids formed during spermiogenesis in two grasshopper species contain expulsed B chromosomes. By using DNA probes for B-specific satellite DNAs in Eumigus monticola and Eyprepocnemis plorans, and also 18S rDNA in the latter species, we were able to count the number of B chromosomes in standard spermatids submitted to fluorescence in situ hybridization, as well as visualizing B chromosomes inside most microspermatids. In E. plorans, the presence of B-carrying microspermatids in 1B males was associated with a significant decrease in the proportion of B-carrying standard spermatids. The fact that this decrease was apparent in elongating spermatids but not in round ones demonstrates that meiosis yields 1:1 proportions of 0B and 1B spermatids and hence that B elimination takes place post-meiotically, i.e., during spermiogenesis, implying a 5-25% decrease in B transmission rate. In E. monticola, the B chromosome is mitotically unstable and B number varies between cells within a same individual. A comparison of B frequency between round and elongating spermatids of a same individual revealed a significant 12.3% decrease. We conclude that B chromosome elimination during spermiogenesis is a defense weapon of the host genome to get rid of parasitic chromosomes.

Satellite DNA content illuminates the ancestry of a supernumerary (B) chromosome.

B chromosomes are supernumerary genomic elements most likely derived from the standard (A) chromosomes, whose dispensability has freed their DNA sequences to evolve fast, thus making it difficult to uncover their ancestry. Here, we show the ancestry of a B chromosome in the grasshopper Eumigus monticola by means of the high-throughput analysis of the satellitome, i.e., the whole collection of satellite DNA (satDNA). The satellitome found in this species consists of 27 satDNA families, with monomer length between 5 and 325 nt and A + T content between 42.9 and 83.3 %. Two out of the 20 clustered satDNA families (EmoSat26-41 and EmoSat27-102) were observed only on the B chromosome. The A chromosome carrying the highest number of satDNA families was the megameric S8 (13 families), six of which were also present in the B chromosome, and three of these were exclusive of the S8 and B chromosomes. The absence in the B chromosome of the H3 histone gene cluster (located interstitially on S8) and three satDNA families (located distally on S8) allowed delimiting the possible origin of the B chromosome to the proximal third of the S8 autosome, through a breakpoint between EmoSat11-122 and the H3 cluster. Interestingly, bioinformatic analysis revealed the presence of seeds for the two B-specific satDNAs in the A chromosomes, suggesting their massive amplification in the B chromosome after its origin. Therefore, intraspecifically arisen B chromosomes can harbor DNA sequences apparently being B-specific.

B-chromosome effects on Hsp70 gene expression does not occur at transcriptional level in the grasshopper Eyprepocnemis plorans.

As intragenomic parasites, B chromosomes can elicit stress in the host genome, thus inducing a response for host adaptation to this kind of continuous parasitism. In the grasshopper Eyprepocnemis plorans, B-chromosome presence has been previously associated with a decrease in the amount of the heat-shock protein 70 (HSP70). To investigate whether this effect is already apparent at transcriptional level, we analyze the expression levels of the Hsp70 gene in gonads and somatic tissues of males and females with and without B chromosomes from two populations, where the predominant B chromosome variants (B2 and B24) exhibit different levels of parasitism, by means of quantitative real-time PCR (qPCR) on complementary DNA (cDNA). The results revealed the absence of significant differences for Hsp70 transcripts associated with B-chromosome presence in virtually all samples. This indicates that the decrease in HSP70 protein levels, formerly reported in this species, may not be a consequence of transcriptional down-regulation of Hsp70 genes, but the result of post-transcriptional regulation. These results will help to design future studies oriented to identifying factors modulating Hsp70 expression, and will also contribute to uncover the biological role of B chromosomes in eukaryotic genomes.

Intragenomic distribution of RTE retroelements suggests intrachromosomal movement.

Much is known about the abundance of transposable elements (TEs) in eukaryotic genomes, but much is still unknown on their behaviour within cells. We employ here a combination of cytological, molecular and genomic approaches providing information on the intragenomic distribution and behaviour of non-long terminal repeat (LTR) retrotransposon-like elements (RTE). We microdissected every chromosome in a single first meiotic metaphase cell of the grasshopper Eyprepocnemis plorans and polymerase chain reaction (PCR) amplified a fragment of the RTE reverse transcriptase gene with specific primers. PCR products were cloned and 139 clones were sequenced. Analysis of molecular variance (AMOVA) showed significant intragenomic structure for these elements, with 4.6 % of molecular variance being found between chromosomes. A maximum likelihood tree built with the RTE sequences revealed the frequent presence of two or more elements showing very high similarity and being located on the same chromosome, thus suggesting intrachromosome movement. The 454 pyrosequencing of genomic DNA gave strong support to the microdissection results and provided evidence for the existence of 5' truncated elements. Our results thus indicate a tendency of RTE elements to reinsert into the same chromosome from where they were transcribed, which could be achieved if retrotranscription and insertion takes place immediately after transcription.

Transient Microgeographic Clines during B Chromosome Invasion.

The near-neutral model of B chromosome evolution predicts that the invasion of a new population should last some tens of generations, but the details on how it proceeds in real populations are mostly unknown. Trying to fill this gap, we analyze here a natural population of the grasshopper Eyprepocnemis plorans at three time points during the last 35 years. Our results show that B chromosome frequency increased significantly during this period and that a cline observed in 1992 had disappeared in 2012 once B chromosome frequency reached an upper limit at all sites sampled. This indicates that, during B chromosome invasion, transient clines for B chromosome frequency are formed at the invasion front on a microgeographic scale. Computer simulation experiments showed that the pattern of change observed for genotypic frequencies is consistent with the existence of B chromosome drive through females and selection against individuals with a high number of B chromosomes.

HP1 knockdown is associated with abnormal condensation of almost all chromatin types in a grasshopper (Eyprepocnemis plorans).

Heterochromatin protein 1 (HP1) is a highly conserved family of eukaryotic proteins required for heterochromatic gene silencing and euchromatic gene transcription regulation. In addition, HP1 is involved in chromatin organization and protection of chromosome integrity during cell division. Here, we present a cytological and molecular analysis of the effects of HP1 knockdown in Eyprepocnemis plorans, a grasshopper species polymorphic for supernumerary heterochromatic chromosomes. Our results revealed contrasting effects of HP1 knockdown on gene activity. While the Bub1 gene decreased in expression level in HP1 knockdown animals, NOR activity, rRNA and, contrarily to previous reports in Drosophila, Hsp70 gene expression remained unchanged. Furthermore, HP1 knockdown resulted in abnormal chromatin condensation, chromosomal bridges, higher frequency of macrospermatids, loss of muscle mass and hemolymph amount as well as a low number of dividing cells and survival reduction. All these phenotypes are very likely due to the chromatin condensation disruption observed for almost all kinds of chromatin.

B chromosomes showing active ribosomal RNA genes contribute insignificant amounts of rRNA in the grasshopper Eyprepocnemis plorans.

The genetic inertness of supernumerary (B) chromosomes has recently been called into question after finding several cases of gene activity on them. The grasshopper Eyprepocnemis plorans harbors B chromosomes containing large amounts of ribosomal DNA (rDNA) units, some of which are eventually active, but the amount of rRNA transcripts contributed by B chromosomes, compared to those of the standard (A) chromosomes, is unknown. Here, we address this question by means of quantitative PCR (qPCR) for two different ITS2 amplicons, one coming from rDNA units located in both A and B chromosomes (ITS2(A+B)) and the other being specific to B chromosomes (ITS2(B)). We analyzed six body parts in nine males showing rDNA expression in their B chromosomes in the testis. Amplification of the ITS2(B) amplicon was successful in RNA extracted from all six body parts analyzed, but showed relative quantification (RQ) values four orders of magnitude lower than those obtained for the ITS(A+B) amplicon. RQ values differed significantly between body parts for the two amplicons, with testis, accessory gland and wing muscle showing threefold higher values than head, gastric cecum and hind leg. We conclude that the level of B-specific rDNA expression is extremely low even in individuals where B chromosome rDNA is not completely silenced. Bearing in mind that B chromosomes carry the largest rDNA cluster in the E. plorans genome, we also infer that the relative contribution of B chromosome rRNA genes to ribosome biogenesis is insignificant, at least in the body parts analyzed.

B chromosomes in the grasshopper Eyprepocnemis plorans are present in all body parts analyzed and show extensive variation for rDNA copy number.

B chromosomes in the grasshopper Eyprepocnemis plorans are considered to be mitotically stable, because all meiotic (primary spermatocytes and oocytes) or mitotic (embryos, ovarioles, and gastric caecum) cells analyzed within the same individual show the same B chromosome number. Nothing is known, however, about body parts with somatic tissues with no mitotic activity in adult individuals, constituting the immense majority of their body. Therefore, we investigated whether B chromosomes are present in 8 non-mitotically active somatic body parts from both sexes in addition to ovarioles and testes by PCR analysis of 2 B-specific molecular markers. We also elucidated the number of B chromosomes that an individual carried through quantifying the B-located rDNA copy number by qPCR. Our results indicated the amplification of both B-specific markers in all analyzed body parts. However, we found high variation between males for the estimated number of rDNA units in the B chromosomes. These results demonstrate the presence of B chromosomes in all body parts from the same individual and suggest a high variation in the rDNA content of the B chromosomes carried by different individuals from the same population, presumably due to unequal crossovers during meiosis.

The Ku70 DNA-repair protein is involved in centromere function in a grasshopper species.

The Ku70 protein is involved in numerous cell functions, the nonhomologous end joining (NHEJ) DNA repair pathway being the best known. Here, we report a novel function for this protein in the grasshopper Eyprepocnemis plorans. We observed the presence of large Ku70 foci on the centromeres of meiotic and mitotic chromosomes during the cell cycle stages showing the highest centromeric activity (i.e., metaphase and anaphase). The fact that colchicine treatment prevented centromeric location of Ku70, suggests a microtubule-dependent centromeric function for Ku70. Likewise, the absence of Ku70 at metaphase-anaphase centromeres from three males whose Ku70 gene had been knocked down using interference RNA, and the dramatic increase in the frequency of polyploid spermatids observed in these males, suggest that the centromeric presence of Ku70 is required for normal cytokinesis in this species. The centromeric function of Ku70 was not observed in 14 other grasshopper and locust species, or in the mouse, thus suggesting that it is an autapomorphy in E. plorans.

Ribosomal DNA is active in different B chromosome variants of the grasshopper Eyprepocnemis plorans.

B chromosomes are considered to be genetically inert elements. However, some of them are able to show nucleolus organizer region (NOR) activity, as detected by both cytological and molecular means. The grasshopper Eyprepocnemis plorans shows a B chromosome polymorphism characterized by the existence of many B variants. One of them, B24, shows NOR activity in about half of B-carrying males in the Torrox population. Molecular data have suggested the recent origin for B chromosomes in this species, and on this basis it would be expected that NOR activity was widespread among the different B variants. Here we test this hypothesis in four different B chromosome variants (B1, B2, B5, and B24) from 11 natural populations of the grasshopper E. plorans covering the south and east of the Iberian Peninsula plus the Balearic Islands. We used two different approaches: (1) the cytological observation of nucleoli attached to the distal region of the B chromosome (where the rDNA is located), and (2) the molecular detection of the rDNA transcripts carrying an adenine insertion characteristic of B chromosome ITS2 sequences. The results showed NOR expression not only for B24 but also for the B1 and B2 variants. However, the level of B-NOR expression in these latter variants, measured by the proportion of cells showing nucleoli attached to the B chromosomes, was much lower than that previously reported for B24. This suggests the possibility that structural or genetic background conditions are enhancing the expressivity of the rDNA in the B24 variant.

Tandem Repeat DNA Provides Many Cytological Markers for Hybrid Zone Analysis in Two Subspecies of the Grasshopper Chorthippus parallelus.

Recent advances in next generation sequencing (NGS) have greatly increased our understanding of non-coding tandem repeat (TR) DNA. Here we show how TR DNA can be useful for the study of hybrid zones (HZ), as it serves as a marker to identify introgression in areas where two biological entities come in contact. We used Illumina libraries to analyse two subspecies of the grasshopper Chorthippus parallelus, which currently form a HZ in the Pyrenees. We retrieved a total of 152 TR sequences, and used fluorescent in situ hybridization (FISH) to map 77 families in purebred individuals from both subspecies. Our analysis revealed 50 TR families that could serve as markers for analysis of this HZ, using FISH. Differential TR bands were unevenly distributed between chromosomes and subspecies. Some of these TR families yielded FISH bands in only one of the subspecies, suggesting the amplification of these TR families after the geographic separation of the subspecies in the Pleistocene. Our cytological analysis of two TR markers along a transect of the Pyrenean hybrid zone showed asymmetrical introgression of one subspecies into the other, consistent with previous findings using other markers. These results demonstrate the reliability of TR-band markers for hybrid zone studies.

Gypsy, RTE and Mariner transposable elements populate Eyprepocnemis plorans genome.

We analyze here the presence and abundance of three types of transposable elements (TEs), i.e. Gypsy, RTE and Mariner, in the genome of the grasshopper Eyprepocnemis plorans. PCR experiments allowed amplification, cloning and sequencing of these elements (EploGypI, EploRTE5, EploMar20) from the E. plorans genome. Fluorescent in situ hybridization (FISH) showed that all three elements are restricted to euchromatic regions, thus being absent from the pericentromeric region of all A chromosomes, which contain a satellite DNA (satDNA) and ribosomal DNA (rDNA), and being very scarce in B chromosomes mostly made up of these two types of repetitive DNA. FISH suggested that EploGypI is the most abundant and EploMar20 is the least abundant, with EploRTE5 showing intermediate abundance. An estimation of copy number, by means of quantitative PCR, showed that EploGypI is, by far, the most abundant element, followed by EploRTE5 and EploMar20, in consistency with FISH results. RNA isolation and PCR experiments on complementary DNA (cDNA) showed the presence of transcripts for the three TE elements. The implications of the preferential location of these TE elements into euchromatin, the significance of TE abundance in the giant genome of this species, and a possible relationship between TEs and B chromosome mutability, are discussed.