RESEARCH ARTICLE Open Access The satellite DNA AflaSAT-1 in the A and B chromosomes of the grasshopper Abracris flavolineata Diogo Milani1, Érica Ramos2, Vilma Loreto3, Dardo Andrea Martí4, Adauto Lima Cardoso2, Karen Cristiane Martinez de Moraes1, Cesar Martins2 and Diogo Cavalcanti Cabral-de-Mello1* Abstract Background: Satellite DNAs (satDNAs) are organized in repetitions directly contiguous to one another, forming long arrays and composing a large portion of eukaryote genomes. These sequences evolve according to the concerted evolution model, and homogenization of repeats is observed at the intragenomic level. Satellite DNAs are the primary component of heterochromatin, located primarily in centromeres and telomeres. Moreover, satDNA enrichment in specific chromosomes has been observed, such as in B chromosomes, that can provide clues about composition, origin and evolution of this chromosome. In this study, we isolated and characterized a satDNA in A and B chromosomes of Abracris flavolineata by integrating cytogenetic, molecular and genomics approaches at intra- and inter-population levels, with the aim to understand the evolution of satDNA and composition of B chromosomes. Results: AflaSAT-1 satDNA was shared with other species and in A. flavolineata, was associated with another satDNA, AflaSAT-2. Chromosomal mapping revealed centromeric blocks variable in size in almost all chromosomes (except pair 11) of A complement for both satDNAs, whereas for B chromosome, only a small centromeric signal occurred. In distinct populations, variable number of AflaSAT-1 chromosomal sites correlated with variability in copy number. Instead of such variability, low sequence diversity was observed in A complement, but monomers from B chromosome were more variable, presenting also exclusive mutations. AflaSAT-1 was transcribed in five tissues of adults in distinct life cycle phases. Conclusions: The sharing of AflaSAT-1 with other species is consistent with the library hypothesis and indicates common origin in a common ancestor; however, AflaSAT-1 was highly amplified in the genome of A. flavolineata. At the population level, homogenization of repeats in distinct populations was documented, but dynamic expansion or elimination of repeats was also observed. Concerning the B chromosome, our data provided new information on the composition in A. flavolineata. Together with previous results, the sequences of heterochromatic nature were not likely highly amplified in the entire B chromosome. Finally, the constitutive transcriptional activity suggests a possible unknown functional role, which should be further investigated. Keywords: B chromosome, Repetitive DNA, Tandem repeat, Transcription * Correspondence: mellodc@rc.unesp.br 1Departamento de Biologia, UNESP - Univ Estadual Paulista, Instituto de Biociências/IB, Rio Claro, São Paulo CEP 13506-900, Brazil Full list of author information is available at the end of the article © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Milani et al. BMC Genetics (2017) 18:81 DOI 10.1186/s12863-017-0548-9 http://crossmark.crossref.org/dialog/?doi=10.1186/s12863-017-0548-9&domain=pdf mailto:mellodc@rc.unesp.br http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/publicdomain/zero/1.0/ Background For more than half a century, researchers have attempted to understand the complexity and evolution of the repeti- tive DNA fraction of the genome. Repetitive DNAs are classified depending on the rate of repetitiveness and ar- rangement in the genome and include Transposable Ele- ments (TEs) and minisatellite, microsatellite and satellite DNA (satDNA) [1, 2]. The satDNAs are a highly repetitive fraction of the genome that are organized by repetitions directly contiguous to one another, in tandem, forming long arrays with hundreds of copies generally composed of 100–1000 base pairs [3]. These in tandem arrays compose most of the content of heterochromatin in eukaryotes, associated generally with the centromeres and telomeres [3–6]. These sequences evolve by the homogenization and fixation of different variants in a determined sexual popula- tion under the process of concerted evolution [7, 8]. Mo- lecular mechanisms of DNA turnover, such as unequal crossing over and gene conversion, are primarily respon- sible for this homogenization pattern, leading to quantita- tive changes between species and generating specific satDNA subfamilies with differential arrangements and organization in a given genome [2, 9, 10]. For satDNA tran- scription, multiple functions are indicated to date [11], such as acting through RNAi as an epigenetic regulator of het- erochromatin [12], playing a role as a structural element of centromeres, such as the alpha satellite in humans [13], and also regulating gene expression in yeast [14]. B chromosomes, which are extra elements to the stand- ard complement (A complement) of some species, occur in approximately 15% of eukaryotes [15–20]. These chromo- somes are known to be dispensable for normal develop- ment, for not recombining with standard A chromosomes and for their potential to present a drive mechanism and for accumulation [21]. Very characteristically, B chromo- somes frequently accumulate repetitive DNAs, including satDNAs. Because of the repetitive DNA content, for a long time, B chromosomes were considered genetically inert [21]. However, studies revealed genes in B chromosomes [22–27] that could be transcriptionally active, such as in Crepis capillaris [28], Capreolus pygargus [29], Eyprepocne- mis plorans [30], and Trichogramma kaykai [31]. Moreover, sequences in B chromosomes can influence expression pat- terns from genes in A chromosomes [32–34]. These data suggest a putative biological role for B chromosomes in some species. Although frequently reported in some groups, informa- tion on satDNA populating B chromosomes in insects is restricted to a few species, such as the grasshoppers Eypre- pocnemis plorans [15, 35, 36] and Eumigus monticola [37], wasps [38, 39], Glossina [40] and Drosophila subsilvestris [41]. In these B chromosomes, a direct association is ob- served between heterochromatin and accumulation of re- petitive DNAs. In the organisms listed above, the isolated satDNAs helped in tracking the origin and evolution of B chromosomes. In the grasshopper Abracris flavolineata with karyotype composed of 2n = 23, ×0 (males), one or two submetacen- tric B chromosomes were detected in individuals from a population sampled in Rio Claro/SP-Brazil [24]. Some studies address B chromosome origin and evolution in this species using repetitive DNAs as markers [24, 42, 43], revealing clues about B chromosome composition and ori- gin. Here, we used restriction enzymatic digestion to iso- late the first satDNA in the species, named AflaSAT-1, with the aim to further understand satDNAs among grass- hoppers at the population level and their contribution to B chromosome structure and evolution in A. flavolineata. Finally, AflaSAT-1 was studied using a combination of chromosomal and molecular analyses at the population level to uncover the genome organization and evolution in A and B chromosomes. Results Isolation and characterization of satDNA sequences The genomic DNA from individuals of the Rio Claro/SP population was digested using three different restriction endonucleases (RE), i.e., HindII, AluI and SmaI. A ladder pattern was revealed for HindII and SmaI enzymes with bands of approximately 150, 300, and 450 bp, corre- sponding to the monomer and multimers of a putative tandem sequence. The monomer generated by HindII was selected for further analysis (Fig. 1a). The monomer generated a sequence of 173 bp, after cloning and se- quencing analysis. A convergent internal primer was de- signed and recovered a PCR fragment containing 137 bp, which formed a ladder pattern, typical for satD- NAs (Fig. 1b, c). This sequence was named AflaSAT-1. Internal restriction sites for other enzymes were also recognized in AflaSAT-1 (Fig. 1c). With PCR, AflaSAT-1 was detected in individuals in six populations that were sampled and in the μB-DNA from the Rio Claro/SP population. A total of 79 AflaSAT-1 sequences were recovered after cloning of in- dividuals from Rio Claro/SP (15 clones), μB-DNA from Rio Claro/SP (10 clones), Cabo/PE (11 clones), Santa Bárbara do Pará/PA (10 clones), Paranaíta/MT (11 clones), Manaus/AM (12 clones), and Posadas/AR (10 clones). The AflaSAT-1 was similar in composition in all populations, with approximately 41.0% G + C base pairs. Different numbers of haplotypes were recognized de- pending on the population. Considering all sequences, 11 haplotypes were recognized. The number of muta- tions and variable sites was slightly higher in the se- quences recovered from μB-DNA than in those from A chromosomes of the six populations. Nucleotide and haplotype diversity was also slightly variable, reaching the highest values in the Cabo/PE population (Table 1). Milani et al. BMC Genetics (2017) 18:81 Page 2 of 11 A minimum spanning tree revealed the relationships among the 11 haplotypes from all populations (Fig. 2). The tree of the 11 haplotypes was shared among at least five populations (Hap1, Hap3 and Hap5). The difference among the haplotypes was only one mutation (substitu- tion), except for the haplotypes from exclusive μB-DNA that were differentiated by three or four mutations, Hap4 and Hap11. Finally, the μB-DNA presented a se- quence grouped to Hap2, which occurred in Cabo/PE and Manaus/AM populations (Fig. 2). Using the sequenced genome from one individual har- boring one B chromosome, we identified one cluster that was similar to AflaSAT-1 in the assembly of RepeatEx- plorer. This cluster represented 1.04% of the genome, corresponding to the fourth most representative se- quence. The cluster retrieved a total of 32 contigs, but only 19 entire sequences of AflaSAT-1 could be recov- ered, in addition to truncated sequences. Unexpectedly, in a few contigs and occurring with AflaSAT-1, a differ- ent sequence containing 242 bp was observed, which was named AflaSAT-2. For this repeat, we recovered ten entire sequences and some truncated repeats. Two ar- rangements were observed in the recovered contigs, only the AflaSAT-1 and AflaSAT-1 plus AflaSAT-2; the AflaSAT-2 was never recovered alone. Chromosome distribution and copy number variation of AflaSAT-1 in populations of A. flavolineata The FISH using AflaSAT-1 and AflaSAT-2 probes in in- dividuals from Rio Claro/SP revealed large pericentro- meric blocks in all chromosomes, with blocks varying in size, except for pair 11 that did not have visible signals (Fig. 3a-d). The B chromosome showed a small signal in the centromeric region (Fig. 3e, f ). These two sequences were interglimed with one another, as observed in fiber- FISH experiments (Fig. 3g). Association of AflaSAT-1 and AflaSAT-2 forming a composed unit was also ob- served in sequences retrieved from sequenced genomes. Analysis of chromosomal distribution of AflaSAT-1 in different populations revealed identical patterns for indi- viduals from Rio Claro/SP and from Sta. Bárbara do Pará/PA (Fig. 4a). In the other populations, a slight vari- ation was observed, i.e., in Cabo/PE, the pair 1 did not have a signal and the pair 11 had a small positive signal (Fig. 4b), whereas individuals from Posadas/AR had sig- nals in all chromosomes (Fig. 4c). Table 1 Polymorphisms of AflaSAT-1 in distinct populations of A. flavolineata and from μB-DNA Rio Claro/SP 0B gDNA Rio Claro/SP μB- DNA Manaus/ AM Paranaíta/ MT Cabo/ PE Sta Bárbara do Pará/ PA Posadas/ AR All Number of sequences 15 10 12 11 11 10 10 79 Number of haplotypes 4 3 5 3 5 3 4 11 Number of sites 137 137 137 137 137 137 137 137 Number of variable sites 4 7 5 3 5 3 4 10 Number of mutations 4 6 5 3 5 3 4 10 G + C proportion 44.5% 44.6% 44.3% 44.3% 45.0% 44.5% 44.5% 44.5% Nucleotide diversity (π) 0.00876 0.01217 0.01615 0.01035 0.01859 0.01071 0.01184 0.01951 Standard deviation (π) 0.00204 0.00725 0.00211 0.00172 0.00187 0.00165 0.00255 0.00141 Haplotypes diversity (Hd) 0.600 0.511 0.833 0.655 0.855 0.689 0.644 0.845 Fig. 1 Agarose gel electrophoresis (a) of integrated genomic DNA (1) and the same sample digested with HindII (2); (b) result of amplification of AflaSAT-1; (c) sequence of an entire monomeric unit of AflaSAT-1. In (a, b), L corresponds to 1 Kb plus marker, and asterisks indicate the monomer, dimer, trimer and tetramer. The arrows in (c) show the internal primers Milani et al. BMC Genetics (2017) 18:81 Page 3 of 11 Additionally, we observed copy number variation of AflaSAT-1 with qPCR. Males without and with one B chromosome from Rio Claro/SP showed differences, with the individuals harboring the B chromosome having a higher copy number, as expected. Females from Rio Claro/SP also had a higher copy number, which was at- tributed to two copies of the X chromosome. Compared with those of Rio Claro/SP, individuals from Cabo/PE had fewer copies, whereas individuals from Santa Bár- bara do Pará/PA had similar copy numbers. Finally, indi- viduals from Posadas/AR had the highest number of copies (Fig. 5). The high standard deviations, considering the absolute copy number quantified, indicated that the copy number of AflaSAT-1 is very unstable and variable individually (Additional file 1). Occurrence of AflaSAT-1 in other grasshopper species In the dot blot test for AflaSAT-1, among the six species tested, none revealed positive signals, and positive hybridization was restricted to genomic DNA from A. flavolineata. In the two species of the Ommatolampidi- nae, the occurrence of AflaSAT-1 was also tested using FISH, and no signals were observed (data not shown). However, with PCR amplification, the AflaSAT-1 mono- mer was detected in A. dilecta, V. rugulosa and R. bergi and further confirmed through sequencing of PCR prod- ucts for each species (Additional file 2). Transcriptional activity of AflaSAT-1 Based on RT-PCR analysis, AflaSAT-1 was transcription- ally active in all different organ tissues tested in male and female adults, with activity also in both embryos and nymphs. The different tissues included the head, sal- tatory leg, testis, ovariole and gastric caecum, in addition to embryos and first stage nymphs. These diverse sam- ples were studied to test for constitutional transcription of AflaSAT-1, which could suggest putative function for the organism. Tissues from somatic and germ line line- ages were also studied to reinforce putative constitu- tional function. The 1B male individuals also showed transcriptional activity for AflaSAT-1. For the analyses of all tissues and life stages, the most evident bands corre- sponded to monomers and tetramers of AflaSAT-1 DNA (Fig. 6), and the sequencing of monomers from ran- domly selected samples confirmed the specific transcrip- tional activity of AflaSAT-1. Discussion AflaSAT-1 organization and evolution in a chromosomes of Abracris flavolineata satDNA families have been described for only 12 grass- hopper species, most members of Acrididae. The repre- sentation of AflaSAT-1 was low (1.04%) in the A. flavolineata genome but corresponded to the fourth most abundant repetitive DNA (Milani et al., unpub- lished data). In grasshoppers, different satDNA repeats commonly represent less than 1% of the genome, as ob- served L. migratoria and E. monticola [37, 44]. However, highly abundant sequences were observed in Schisto- cerca gregaria with different satDNA families represent- ing approximately 18% (SG1) and 12% (SG2-alfa) [45]. As expected, the general chromosomal distribution of AflaSAT-1 was coincident with pericentromeric hetero- chromatin, a common pattern among eukaryotes and Fig. 2 Haplotype network for the 11 haplotypes and their relationships from sequences of AflaSAT-1 obtained from different populations. Black dots correspond to substitutions, and the haplotype circle diameter corresponds to abundance. The different populations are represented in different colors Milani et al. BMC Genetics (2017) 18:81 Page 4 of 11 observed extensively in grasshoppers [35, 37, 44–46]. The occurrence of another satDNA, i.e., AflaSAT-2, with the same distribution as that of AflaSAT-1 indicated a more complex structure, which was demonstrated by the two types of arrangement in the A. flavolineata gen- ome. This arrangement is similar to that described in Drosophila buzzatii for pBuM-1 and pBuM-2 satDNA subfamilies [47]. The similar distributions of non- homologous satDNAs observed in this study have also been reported in some Drosophila [48], rye [49] and ro- dents [50]. Based on interspecies analysis, AflaSAT-1 was not spe- cific to A. flavolineata and was recovered from the ge- nomes of three other species with PCR, although dot blot analysis did not reveal positive hybridization. Al- though not species-specific, the copy number of AflaSAT-1 in the genomes of A. dilecta, V. rugulosa and R. bergi is probably low, whereas AflaSAT-1 was highly amplified in the genome of A. flavolineata, generating a clustered organization. Divergence among sequences among the different species was discarded as an explan- ation for non-positive dot blot signals, because the se- quencing of PCR products revealed almost no mutations among sequences. The sharing of AflaSAT-1 among dif- ferent species is consistent with the “library hypothesis” by which related species share satDNA families that can be stochastically amplified or not in the diverging ge- nomes [51]. Considering R. bergi, as a member of Mela- noplinae, and A. flavolineata, A. dilecta and V. rugulosa, as members of Ommatolampidinae and the sharing of AflaSAT-1, the origin of this satDNA likely occurred in a common ancestor before the divergence of the two sub- families, which remits to approximately less than 73 mya, corresponding to the time of origin of Acrididae [52]. Re- cently, the most comprehensive comparison between the satellitomes of two other grasshopper species, i.e., L. migratoria (Acrididae) and E. monticola (Pamphagidae), showed that the life span of a satDNA library is less than approximately 100 mya, corresponding to the most recent common ancestor between the two families [37]. Our re- sult was within this life span and showed that the diver- gence between satDNA libraries is variable in Acrididae, considering that the AflaSAT-1 was found in some spe- cies, depending on the subfamily, but not in others. Low variability was observed for AflaSAT-1, and add- itionally, almost no distinct or population-specific profile was observed, which has also been documented for other species consistent with the concerted evolution model for homogenization of repeats at the intrage- nomic level [53–57]. By contrast, chromosomal variabil- ity was observed for cluster number, size, distribution and abundance of repeats, depending on the population. These data indicate that a random expansion or elimin- ation of the satDNA may occur, generating different Fig. 4 Chromosomal location of AflaSAT-1 in three different popula- tions of A. flavolineata. (a) Sta Bárbara do Pará/PA, (b) Cabo/PE and (c) Posadas/AR. The X chromosome is indicated, and the arrows point to the pair 11. In (b), the largest chromosome, chromosome 1, is also indicated Fig. 3 FISH in mitotic embryo chromosomes (a-f) and in distended chromatin fiber (g) of AflaSAT-1 and AflaSAT-2 satDNAs in individuals without (a-d, g) and with one B chromosome (e, f). Arrows in (c, e, f) show the pair 11 and the arrowhead in (e, f) the B chromosome. In (e, f), the B chromosome is highlighted in an inset. Each probe is indicated directly in the panel. Bar = 5 μm Milani et al. BMC Genetics (2017) 18:81 Page 5 of 11 patterns in different populations. Variation related to satDNA mapping was also found among populations of the frog Physalaemus cuvieri [58] and in the grasshopper Eyprepocnemis plorans [59], suggesting a high rate of amplification or deletion mutational events. As expected, the population-dependent higher or lower number of chromosomal clusters for AflaSAT-1 was also consistent with the increase or decrease of AflaSAT-1 copy number, corroborating the dynamic of amplification and deletion of repeats. This change in copy number could be in- duced by several events, such as unequal crossing-over, slippage replication, extrachromosomal circular DNA and rolling-circle, which are common for repetitive DNAs [3, 60, 61]. AflaSAT-1 and its relationship with the B chromosome To date, satDNA in B chromosomes has been reported in few species of grasshoppers, such as Eyprepocnemis plorans [15] and Eimugus monticola [37], which revealed important characteristics about the origin and evolution of B chromosomes. In A. flavolineata, AflaSAT-1 did not disclose clues concerning the specific origin of the B chromosome because of the occurrence in almost all centromeres of A complement. However, AflaSAT-1 helped to understand the composition and molecular evolution of the B chromosome. The C-positive hetero- chromatin blocks are absent in the B chromosome of A. flavolineata [24, 42], and in the chromosomal arms, dif- ferent sequences are shared with the euchromatic Fig. 5 Relative quantification of AflaSAT-1 from different populations of A. flavolineata in comparison with Rio Claro/SP 0B male individuals. (1) Rio Claro/SP male 0B, (2) Rio Claro/SP male 1B, (3) Rio Claro/SP female 0B, (4) Cabo/PE male 0B, (5) Sta Bárbara do Pará/PA male 0B, (6) Posadas/AR male 0B Fig. 6 RT-PCR electrophoresis of AflaSAT-1 using as template cDNA obtained from different tissues, nymphs and embryos. he = head, gc = gastric caecum, tes = testis, ov = ovariole, leg = saltatory leg, nym = nymph, emb = embryo, C- = negative control Milani et al. BMC Genetics (2017) 18:81 Page 6 of 11 regions of A complement. It is considered remarkable for mobile elements and anonymous sequences to be ob- tained from a microdissected B chromosome [43, 61]. The heterochromatin-enriched sequences of A comple- ment that occur in the B chromosome are restricted to a small signal in the centromeric region, as described in this study for AflaSAT-1 and for the C0t-1 DNA [24]. Together, these data suggest that the heterochromatic sequences were not highly amplified in the B chromo- some of A. flavolineata, as is frequently observed in other species [18], and that this chromosome most likely bears unknown euchromatic sequences. Hence, genes and other satDNAs should be investigated for a clearer picture regarding this scenario. Molecular sequence analysis of AflaSAT-1 recovered from the microdissected B chromosome revealed exclu- sive mutations, with a higher number of variable sites and mutations in this chromosome. The higher sequence variability of B chromosome than that of A complement is commonly reported and has been attributed to the higher tolerance of mutations because of the dispensable nature of chromosome B [17]. Similar data are also ob- served for other types of repetitive DNAs, such as his- tone genes and 18S rRNA gene in the grasshopper L. migratoria [62] and in the fish Astyanax paranae [63]. However, in E. plorans, two different sequences (satDNA and 45S rDNA), and in A. flavolineata, the U2 snDNA, revealed different homogenization patterns with high similarity between A and B chromosomes [61, 64]. The discrepancy in sequence variability observed for the two repetitive DNAs in the B chromosome of A. flavolineata, i.e., AflaSAT-1 and U2 snDNA, suggests that different sequences with variable roles have divergent evolution- ary patterns. AflaSAT-1 is transcriptionally active satellite sequence SatDNA transcripts have been reported in different eu- karyotes, and the evidence is accumulating that satellite transcription might be a common placement with putative structural or functional role for genomes [10]. Included among the important roles of satDNAs are heterochroma- tin formation and regulation, involvement in centromere function, epigenetic chromatin silencing and modulation, and regulation of genes, among others [65]. Among insects, satDNA transcription has been de- scribed generally in Hymenoptera, Orthoptera, Diptera and Coleoptera [66] and in the lepidopteran species Cydia pomonella [67] and Plodia interpunctella [68]. Differential transcription of satDNAs in this group is re- lated to different developmental stages and tissues and also the sexes [54, 69, 70]. Considering this information and the constitutional transcription of AflaSAt-1, we suggest an unknown biological function for AflaSAT-1 satDNA, such as a regulatory element, with a structural or functional role, in the A. flavolineata genome. In fu- ture research, the quantitative differential transcription of AflaSAT-1 in A. flavolineata, depending on tissue, sex and presence/absence of B chromosome, must be deter- mined to obtain more precise information for putative functional roles. Methods Animal sampling A total of 129 adult A. flavolineata were collected at five different sites in Brazil (BR) and one from Argentina (AR): Rio Claro/SP (São Paulo), 22°24′45″ S, 47°31′28″ W (30 individuals); Cabo/PE (Pernambuco), 8°17′15″ S, 35°2′7″ W (19 individuals); Sta. Bárbara do Pará/PA (Pará), 1°13′27″ S, 48°17′38″ W (60 individuals); Para- naíta/MT (Mato Grosso), 9°40′25″ S, 56°28′36″ W (5 individuals); Manaus/AM (Amazonas), 3°06′02″ S, 59° 58′31″ W (4 individuals); and Posadas/Misiones/AR, 27o25’ S, 55o56’ W (14 individuals). Testes were fixed in 3:1 absolute ethanol:acetic acid and stored at −20 °C. En- tire animals were immersed in absolute ethanol and stored at −20 °C for genomic DNA (gDNA) extraction. We obtained tissues for DNA extraction and for chro- mosomes from Rio Claro/SP, Cabo/PE, Sta Bárbara do Pará/PA and Posadas/AR populations, whereas from Manaus/AM and Paranaíta/MT populations, only tissues for DNA extraction were obtained. For each analysis, at least three individuals were used. To obtain embryos, animals collected in Rio Claro/SP were placed in plastic boxes until oviposition. Embryo mitotic cells were obtained from embryo dissection approximately 15 days after deposition, following [71] method. Restriction enzymatic digestion, cloning and primer design The genomic DNA was extracted using the phenol- chloroform method [72] and stored at −20 °C until use. Genomic DNA of the specimens from the Rio Claro/SP population was fragmented by restriction enzymatic di- gestion using HindII, AluI and SmaI enzymes. The digested products were fractionated by electrophoresis in 1% agarose gel to verify a ladder pattern, typical for in tandem DNA sequences. Fragments with a ladder pat- tern were selected and purified using a Zymoclean™ Gel DNA Recovery Kit (Zymo Resarch Corp., The Epigenet- ics Company, USA) according to the manufacturer’s instructions. The purified products were cloned using a pMOSBlue Blunt Ended Cloning Kit (GE Healthcare) with DH5α Escherichia coli as competent cells. Positive colonies were randomly chosen and screened by Polymerase Chain Reac- tion (PCR) using M13 primers set (F-5’GTAAAAC- GACGGCCAG and R-5’CAGGAAACAGCTATGAC) for Milani et al. BMC Genetics (2017) 18:81 Page 7 of 11 DNA sequencing by Macrogen Inc. (Korea). Geneious v4.8.5 software [73] was used to check quality and ex- clude vector sequences. To amplify the satDNA se- quence (AflaSAT-1) using PCR, the specific primer set (F-5’GACAGTTTTAAACACTTCCATTACAG and R- 5’GACTGTGTTGATATCCAATAACA)was designated. PCR amplification and sequence analysis Using specific primers for AflaSAT-1, PCR amplification was conducted using the genomic DNA as template of ani- mals from the six different populations and from the B chromosome previously microdissected (μB-DNA) from individuals from Rio Claro/SP [61]. PCR products were vi- sualized on a 1% agarose gel, and the bands were isolated and purified using a ZymocleanTM Gel DNA Recovery Kit (Zymo Research Corp., The Epigenetics Company, USA) according to the producer’s recommendations. For cloning of the purified PCR products, pGEM-T easy vector (Promega, Madison, WI, USA) was used with DH5α Escherichia coli as competent cells. Positive clones were screened using the M13 primers and sequenced by Macrogen Inc. (Korea). The monomer sequences were analyzed using Geneious v4.8.5 [73], and then, DNA polymorphism and haplotype recognition were checked using the DnaSP v.5.10.01 tool [74]. For more accurate analysis, singleton sequences were discarded. A graphical haplotype network was constructed employing Network 4.6.1.2 software (http://www.fluxus- engineering.com). The correspondent haplotype sequences were deposited in NCBI database under the accession numbers MF752447-MF752457. We searched the AflaSAT-1 repeats in the sequenced genome of A. flavolineata from an individual harboring one B chromosome. This genome was sequenced using Illumina Miseq paired-end (2 × 300), and the libraries were constructed using a Nextera DNA Library Prepar- ation Kit and quantified by a KAPA Library Quantifica- tion Kit (Milani et al., in preparation). For these purposes, we applied the graph-based clustering and as- sembly using RepeatExplorer [75] and manually searched for sequences similar to AflaSAT-1 in the assembled contigs using Geneious v4.8.5 [73]. For this search, we considered only clusters showing high graph density that are typical for satDNA [76]. In the clusters containing the AflaSAT-1, we also observed another associated se- quence (named AflaSAT-2), and a primer set to recover this sequence was designed, F-5’GGGTCTCGCGAAAT- GAGAC and R-5’GCTTTCTAAACGGAATCGAG. Chromosome obtaining and fluorescent in situ hybridization (FISH) Meiotic cells were obtained from testes, whereas mitotic cells were obtained from embryos. For conventional ana- lyses used to check the general chromosomal structure of animals from different populations, the slides were prepared with tissue maceration and staining with Giemsa 5%. The PCR products from satDNAs from Rio Claro/SP individuals were used as probes for FISH assays using chromosomes of individuals from the populations of Rio Claro/SP, Cabo/PE, Sta Bárbara do Pará/PA and Posadas/ AR. Fragments were labeled by nick translation using digoxigenin-11-dUTP and detected by anti-digoxigenin rhodamine (Roche, Mannheim, Germany) or biotin-14- dUTP detected with streptavidin Alexa Fluor-488 conju- gated (Invitrogen, San Diego, CA, USA). FISH experiments were conducted following [77], with adaptations by [78]. Fiber-FISH was conducted according to [79]. Slides were counterstained with DAPI (4′,6-Diamidine-2′-phenylin- dole) and mounted with VECTASHIELD (Vector, Burlin- game, CA, USA). Pictures were captured using a DP70 cooled digital camera in gray scale coupled with an Olym- pus microscope BX51 equipped with a fluorescence lamp and appropriate filters. The images were pseudo-colored, merged and treated for brightness and contrast using Adobe Photoshop CS6. Dot blot hybridization To determine whether the AflaSAT-1 was shared with other species at the level of genus, subfamily and family, we conducted a dot blot experiment, following the de- scriptions from [80], using the sequence amplified from individuals from Rio Claro/SP as a probe for the other six species. The probe was tested against the genomic DNA of A. flavolineata as positive control, the congen- eric species A. dilecta and one another species in the same subfamily (Ommatolampidinae), i.e., Vilerna rugu- losa. Moreover, we used species from other subfamilies of Acrididae, including Melanoplinae (Ronderosia bergi), Cyrtacanthacridinae (Schistocerca pallens), Gomphoceri- nae (Amblytropidia robusta) and Leptysminae (Eumas- tusia koebelei koebelei). The presence of AflaSAT-1 was also tested using PCR in each species following the same conditions applied for A. flavolineata described above, and the results were checked by nucleotide sequencing. Copy number detection by qPCR To verify the copy number of AflaSAT-1 among differ- ent populations and in individuals harboring one B chromosome, we performed a relative quantification employing the gene dose ΔCt method [81]. qPCR assays were performed using the gDNA from male samples of each population for which we obtained FISH results, i.e., Cabo/PE, Sta Bárbara do Pará/PA and Posadas/AR. For the Rio Claro/SP population, we tested males with and without one B chromosome and females without a B chromosome. The gene dosage ratios (GDR) were ob- tained following the same parameter of [25], using a Milani et al. BMC Genetics (2017) 18:81 Page 8 of 11 http://www.fluxus-engineering.com http://www.fluxus-engineering.com Heat Shock Protein (Hsp70) as the reference gene (F- 5’GGTGTGATGACCACTCTTATCAA and R- 5’CACTTCAATTTGAGGCACACC), because no differ- ence was detected in amplification ratio among males, females and 0B and 1B individuals. Our results support that the Hsp70 gene is placed in autosomes, considering no difference between males and females. The target and reference gene were analyzed simultaneously in dupli- cates of four independent samples, and the qPCR condi- tions were set at 95 °C for 10 min; 45 cycles of 95 °C for 15 s, and 60 °C for 1 min, performed in a StepOne Real- Time PCR System (Life Technologies, Carlsbad, CA). Specificity of the PCR products was confirmed by ana- lysis of the dissociation curve. RNA extraction and transcriptional analyses The RNA was extracted from organ tissues of male and female adults, including head, saltatory leg, testis, ovari- ole and gastric caecum, and from embryos (with 20 days of development) and first stage nymphs (immediately after hatch) from the Rio Claro/SP population. We also tested these tissues from adult males with 1B. RNA was extracted using TRIzol® Reagent (Life Technologies) and then treated with Amplification Grade DNAse I (Sigma- Aldrich) to avoid DNA contaminants. Finally, the cDNA samples were obtained by reverse transcription (RT- PCR) using a High-Capacity cDNA Reverse Transcrip- tion Kit (Life Technologies) with the reaction conditions set to 25 °C for 10 min; 37 °C for 120 min, and 85 °C for 5 min. Afterward, the cDNA samples were used as tem- plates for conventional PCR amplification of AflaSAT-1, with at least three biological replicates. To validate the signals of transcription and to ensure that the samples were not contaminated with genomic DNA, the cDNA samples were used as source for ampli- fication of Hsp70 as a control. The amplification of the Hsp70 gene in genomic DNA samples and uncontamin- ated cDNA samples revealed bands with different sizes of approximately 300 and 200 bp, respectively. An intron in the genomic DNA that was not present after RNA transcription and processing caused the difference. Some PCR products were sequenced to confirm the HSP70 sequence. Additional files Additional file 1: qPCR data of gDNA used to calculate the sequence dose by Ct method of relative quantification (Nguyen et al. 2013). Gene dosage ratios (GDR) of the target genes were compared with autosomal gene Hsp70. (XLSX 90 kb) Additional file 2: (a) Dot blot analysis, (b) PCR electrophoresis of AflaSAT-1 and (c) Nucleotide alignment of positively amplified AflaSAT-1 fragments. Af = Abracris flavolineata, 1 = A. dilecta, 2 = Vilerna rugulosa, 3 = Ronderosia bergi, 4 = Schistocerca pallens, 5 = Amblytropidia robusta, 6 = Eumastusia koebelei koebelei. (JPEG 363 kb) Abbreviations 2n: Diploid number; Bp: Base pairs; DAPI: 4′, 6-Diamidino-2-phenylindole; FISH: Fluorescence in situ hybridization; PCR: Polymerase chain reaction; satDNA: Satellite DNA Acknowledgements This study was partially supported by the Fundação de Amparo a Pesquisa do Estado de São Paulo-FAPESP (process number 2011/19481-3, 2015/16661- 1) and Coordenadoria de Aperfeiçoamento de Pessoal de Nível Superior- CAPES. DM was supported by FAPESP (process number 2015/05246-3). The authors are grateful to Dr. Thiago Gazoni for sampling in Manaus/AM, Para- naíta/MT and to the administration of Parque Estadual Edmundo Navarro de Andrade for collecting authorization. DCCM was the recipient of a research productivity fellowship from the Conselho Nacional de Desenvolvimento Ci- entífico e Tecnológico-CNPq (process number 304758/2014-0). Authors’ contributions DM conducted the bioinformatics analysis, qPCR analysis, the chromosome preparations and the molecular cytogenetic experiments, interpreted the data, and drafted of the manuscript. DAM and VL collected animals and interpreted the data and drafted the manuscript. AC and KCMM assisted in qPCR experiments interpreted the data and drafted the manuscript. CM and ER assisted in genome sequencing and bioinformatics analysis and interpreted the data and drafted the manuscript. DCCM conceived the study, participated in its design and coordination, interpreted the data and assisted in drafting the manuscript. All authors have read and approved the final manuscript. Ethics approval and consent to participate Not Applicable. Competing interests The authors declare that they have no competing interests. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Author details 1Departamento de Biologia, UNESP - Univ Estadual Paulista, Instituto de Biociências/IB, Rio Claro, São Paulo CEP 13506-900, Brazil. 2Departamento de Morfologia, UNESP - Univ Estadual Paulista, Instituto de Biociências/IB, Botucatu, São Paulo, Brazil. 3Departamento de Genética, UFPE - Univ Federal de Pernambuco, Centro de Biociências/CB, Recife, Pernambuco, Brazil. 4IBS - UNaM – CONICET, Posadas, Misiones, Argentina. Received: 21 April 2017 Accepted: 22 August 2017 References 1. Biémont C, Vieira C. Genetics: junk DNA as an evolutionary force. Nature. 2006;443:521–4. 2. Plohl M, Meštrović N, Mravinac B. Satellite DNA evolution. In: Garrido-Ramos MA, editor. Repetitive DNA. Genome dynamics. 7th ed. Basel: Karger; 2012. p. 126–52. 3. Charlesworth B, Sniegowski P, Stephan W. The evolutionary dynamics of repetitive DNA in eukaryotes. 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Proc Natl Acad Sci U S A. 2013;110:6931–6. • We accept pre-submission inquiries • Our selector tool helps you to find the most relevant journal • We provide round the clock customer support • Convenient online submission • Thorough peer review • Inclusion in PubMed and all major indexing services • Maximum visibility for your research Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and we will help you at every step: Milani et al. BMC Genetics (2017) 18:81 Page 11 of 11 http://www.geneious.com Abstract Background Results Conclusions Background Results Isolation and characterization of satDNA sequences Chromosome distribution and copy number variation of AflaSAT-1 in populations of A. flavolineata Occurrence of AflaSAT-1 in other grasshopper species Transcriptional activity of AflaSAT-1 Discussion AflaSAT-1 organization and evolution in a chromosomes of Abracris flavolineata AflaSAT-1 and its relationship with the B chromosome AflaSAT-1 is transcriptionally active satellite sequence Methods Animal sampling Restriction enzymatic digestion, cloning and primer design PCR amplification and sequence analysis Chromosome obtaining and fluorescent in situ hybridization (FISH) Dot blot hybridization Copy number detection by qPCR RNA extraction and transcriptional analyses Additional files Abbreviations Authors’ contributions Ethics approval and consent to participate Competing interests Publisher’s Note Author details References