Application of multicolor banding combined with heterochromatic and locus-specific probes identify evolutionary conserved breakpoints in Hylobates pileatus

Background The question what makes Homo sapiens sapiens (HSA) special among other species is one of the basic questions of mankind. A small contribution to answer this question is to study the chromosomal constitution of HSA compared to other, closely related species. In order to check the types and extent of evolutionary conserved breakpoints we studied here for the first time the chromosomes of Hylobates pileatus (HPI) compared to HSA and Hylobates lar (HLA) by means of molecular cytogenetics. Results Overall, 68 new evolutionary conserved breakpoints compared to HSA could be characterized in this study. Interestingly, only seven of those were different compared to HLA. However, application of heterochromatic human DNA-probes provided evidence that observed high chromosomal rearrangement rates of gibbons in HPI happened rather in these repetitive elements than in euchromatin, even though most centromeric positions were preserved in HPI compared to HSA. Conclusion Understanding genomes of other species and comparing them to HSA needs full karyotypic and high resolution genomic data to approach both: euchromatic and heterochromatic regions of the studied chromosome-content. This study provides full karyotypic data and previously not available data on heterochromatin-syntenies of HPI and HSA. Electronic supplementary material The online version of this article (doi:10.1186/s13039-016-0228-x) contains supplementary material, which is available to authorized users.


Background
Understanding evolution of human (Homo sapiens sapiens, HSA) is one of the major interests of man, latest since Charles Darwin published 'The origin of species by means of natural selection' [1]. As Hominoidea superfamily includes great apes (chimpanzees, bonobos, orangutans, gorillas, humans) as well as lesser apes (gibbons; Hylobatidae), studies on chromosomal evolution should include all groups of the superfamily HSA belongs to. For lesser apes it has been shown previously that they underwent high numbers of chromosomal rearrangements including inversions, translocations, fissions and fusions [2] after they divided from their common ancestor with great apes around 15-20 million years ago [3,4]. The chromosomal rearrangement rate of gibbons was suggested to be at least an order of magnitude higher than the average rearrangement rate in mammals [5,6], making them an interesting model of evolution.
The pileated gibbon (HPI) has 44 chromosomes and was first karyotyped in 2007 [17]. Yet, detailed molecular cytogenetic characterization was not done, even though high throughput sequencing of its genome was performed recently [11]. Here we report the characterization of 68 evolutionary conserved breakpoints (ECBs) in HPI based on FISH applying high resolution multicolor banding (MCB), locus-specific probes and also all human repetitive probes apart from centromeric ones. The obtained data is an important addition to the already available molecular data.

Results
Results of the present study are summarized in Tables 1  and 2. Representative results of MCB, as well as regions being homologous to acrocentric short arms in HSA, the NOR-region, the region 9p12/9q13 and 16q11.2 are shown in Fig. 1. Also one locus-specific probe based FISH result for a region being homologous in HPI to human chromosome 2 can be found there. The latter is shown, as this region/rearrangement could not be visualized by MCB.
As shown in Table 1, most HPI chromosomes are composed from regions being homologous to two or more HSA chromosomes; only HPI chromosomes X, Y, 14, 17 and 20 are homologous to only one human chromosome, each. Of those only chromosomes X, Y and 20 do not present gross chromosomal rearrangements according to FISH.  Application of all human repetitive probes for heterochromatic regions (apart from centromeric ones) revealed that there were no homologies present for sequences derived from human Yq12, 1q12, 9q12, and 15p12~11.2. However, the same heterochromatic sequences being present at HSA 16q11.2 (midi71) were detected at HPI 5, those in HSA 9p12/9q13 (midi36) as one block on HPI 9, those in HSA 19p12~19q12 as one block on HPI 13, and those in human acrocentric short arms (midi54) on HPI 3, 8, 12, 17, 19, 21. NOR specific signals were only obtained as one block on both homologues of HPI 15. Finally, a block of HLA specific heterchromatin was present at end of HLA 3p.
The centromeric positions could be determined for all HPI chromosomes (Table 1). Interestingly, 20/23 positions are the same as in HSA. Centromeric positions of HSA chromosomes 11 and 18 are present at HPI 6 centromere. At HPI chromosomes 7 and 14 neocentromers are formed.

Discussion
Here the first molecular cytogenetic study in HPI is presented using FISH-banding, locus-specific and human heterochromatic probes. It could be confirmed that MCB is well suited to gain a genome wide view on ECBs rapidly also in a yet little studied species (for review on comparable studies see [18]) (Fig. 1). Further narrowing down of ECBs can then be easily done using selected locus-specific probes (Tables 1 and 2).
In general, HPI has, compared to human, a highly rearranged karyotype. However, considering changes like translocations and inversions in HLA, chromosomes of HPI are therefore less affected (see Table 2 and [8]). On the other hand heterochromatic regions underwent in HPI a much faster evolution than in HLA; direct comparison is only possible for the regions being present in HSA as acrocentric short arms (midi54-probe) [19]: on the one hand midi54-positive signals are detectable in HLA only at one spot, in HPI at 6 different locations. On the other hand in HLA this DNA amplified at a region homologous to HSA 2, in HPI midi54-specific DNA seeded at regions homologous to centromeric positions of HSA chromosomes 4, 5, and all acrocentric short arms apart from HSA 21p. As homologous regions to HSA 15 and 21 are together on HPI chromosome 12, this also could be due to a fusion of both midi54-positive regions before an inversion in this chromosome took place. At least 57/68 ECBs detected in HPI here are homologous to such detected in HLA. As 4/11 "new ECBs" compared to HLA are in terminal regions of HSA acrocentric short arms (Table 2), regions not present in HLA there, this may reduce the number of ECBs being really different in HPI and HLA to seven within the euchromatic sequences of HPI.
In this study the results obtained from application of heterochromatic probes suggest that chromosomal evolution in HPI was concentrated to these genomics regions, while in HLA euchromatin was hit more frequently. Both events might have been triggered by the recently suggested "propensity for a gibbon-specific retrotransposon (LAVA) to insert into chromosome segregation genes and alter transcription by providing a premature termination site, suggesting a possible molecular mechanism for the genome plasticity of the gibbon lineage" [8]. This, and the fact that centromeric positions changed in 13/23 chromosomes in HLA [19] and only in 3/23 in HPI needs to be elaborated by future studies.

Conclusions
Overall, this study highlights that to study and understand genomes of other species, e.g. in comparison to human, it is necessary first to have the karyotypic data Arrows highlight interchromosomal rearrangements. Abbreviations: midi = microdissection derived probe (consecutively numbered acc. to production or localization); NOR = nucleolus organizer region and then to combine this with more sophisticated ones, like next generation sequencing data. The latter approach is due to technical restrictions principally not able to detect the peculiarities of heterochromatic regions, while molecular cytogenetic approaches cannot provide a basepair-resolution. Thus, only the combination of all available technical means will help us to understand the miracles of evolution and nature in the end.

Cell culture and chromosomal preparation
Immortalized lymphoblast cell lines derived from a male HPI, was provided by the Khon Kaen University, Thailand. Culture techniques as well as chromosome preparation followed standard protocols.

Fluorescence in situ hybridization
All protocols for FISH have been provided in our previous study by us in Fan et al. [18]; details on MCB, single and dual-color FISH techniques which were performed for the applied bacterial artificial chromosome (BAC-) probes and the commercially derived subtelomeric probes can be found there. All here used BAC-probes are listed in Additional file 1: Table S1. The homemade heterochromatin mix (HCM-) probe set [20] was also described there [18] covering chromosomal regions 1q12, 16q11.2 (midi71), 9q12, 9p12/ 9q13 (midi36) 15p11.2-p11.1, all acrocentric short arms (midi54), 19p12/q12 and Yq12.