Karyotypes and recombination patterns of the Common Swift (Apus apus Linnaeus,1758) and Eurasian Hobby (Falco subbuteo Linnaeus, 1758)

Background

Meiotic recombination is the main source of genetic variability in any population of sexually reproducing organisms.For this reason, the number of recombination events (crossovers) per genome (recombination rate) and their distribution along the chromosomes are considered as important genomic characteristics of species and studied actively in plants, fungi and animals (mostly mammals). These studies demonstrated substantial interspecies variation in recombination rate (Dumont and Payseur 2008; Frohlich et al. 2015;Dapper and Payseur 2017; Stapley et al. 2017) while withinpopulation variation was found to be of the same magnitude across various taxa (Ritz et al. 2017). The minimum possible rate of recombination is constrained by the necessity of at least one crossover per pair of homologous chromosomes to ensure their orderly segregation in thefirst meiotic division.Thus, the recombination rate cannot be lower than the haploid chromosome number (n). Studies on mammals demonstrated that chromosome number (2n) and chromosome arm number (FN) are the best predictors of the recombination rate (Pardo-Manuel de Villena and de Sapienza 2001; Segura et al. 2013; Capilla et al. 2016). A well-known phenomenon of crossover interference (a low probability of crossovers to occur close to each other) is an important constraint of the maximum possible rate of recombination(Berchowitz and Copenhaver 2010; Segura et al. 2013). The smaller the chromosome, the less likely it contains more than one crossover. Thus, the total genome size and the total length of the synaptonemal complex (SC, the core structure of pachytene chromosomes) may also serve as predictors of species-specific recombination rate (Peterson et al. 1994;Kleckner et al. 2003). While molecular and cellular mechanisms controlling the recombination rate are more or less clear, the adaptive importance of interspecies differences for this trait remains a matter of discussion (Stapley et al. 2017;Dapper and Payseur 2017; Ritz et al. 2017).

Birds provide a good model to study the evolution of recombination. With rather a small genome size (about 1.4 pg) (Wright et al. 2014) and very conservative karyotypes (2n being about 78–82 and FN being 80–90 in most species) (Griffn and Burt 2014), they have undergone rapid speciation and evolved various adaptations to a wide variety of habitats. Unfortunately, only less than 10%of bird species have been karyotyped (Griffn and Burt 2014). Recombination traits have been studied in as few as nine species by cytological methods (see Table 1 and references therein) and in nine species by linkage analysis(Dawson et al. 2007; Groenen et al. 2009; Jaari et al. 2009;Aslam et al. 2010; Hansson et al. 2010; Kawakami et al.2014; van Oers et al. 2014).

Most of the birds species studied for recombination rate have common karyotypes (2n = 78–82). Only two species with reduced chromosome number (2n = 68 and 74) have been examined and they did not show a drastic reduction of the recombination rate (Lisachov et al.2017a). More birds with atypical karyotype as Apodidae(swifts), Psittaciformes (parrots) and Falconidae (falcons)need to be studied to reveal the cytogenetic and evolutionary basis of interspecies variation in recombination.

There are two reasons why bird meiotic chromosomes are poorly studied. First, the pachytene cells suitable for analysis can only be obtained in short time windows:from testes of adult males during or shortly before the breeding season or from ovaries of female embryos before hatching or chicks soon after hatching (Scanes 2014). Secondly, the bird has to be sacrificed.

In this study, we collected materials from fatally injured Common Swift (Apus apus Linnaeus, 1758) and Eurasian Hobby (Falco subbuteo Linnaeus, 1758) males provided by the local Bird of Prey Rehabilitation Centre. These specimens represent two bird families, which show a wide karyotypic variation. The karyotype of the Common Swift remains unknown. However, the other swifts examined have reduced and variable diploid numbers (from 62 to 70)(Yadav et al. 1995; Torres et al. 2004). Falconids show a very wide variation in diploid chromosome numbers (from 40 to 92) (Christidis 1990). Eurasian Hobby has 2n = 50 (Christidis 1990; Wang and Chen 1998; Nishida et al. 2008).

In this study, we for thefirst time describe the somatic and pachytene karyotypes of the male Common Swift and the pachytene karyotype of the male Eurasian Hobby. We estimate the overall number and distribution of recombination events along the chromosomes of these species, using immunolocalization of SYCP3, the protein of the SC lateral elements, MLH1, the mismatch repair protein marking mature recombination nodules, and centromere proteins.This method has been successfully used to analyze recombination landscapes infish (Moens 2006; Lisachov et al.2015), reptiles (Lisachov et al. 2017b, c), mammals (Capilla et al. 2016) and birds (Pigozzi 2016). This approach has lower resolution than linkage analysis at thefine scale level.However, cytological approach provides more reliable estimate of total number of global recombination rate, because it does not depend on the number and location of informative markers. Immunocytological analysis of SC spreads is especially effcient to examine bird karyotypes, because it provides unambiguous visualization of all microchromosomes, while at conventional and differential stained metaphase spreads it is diffcult to distinguish microchromosomes from non-specifically stained debris.

Methods

Specimens

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The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Table 1 Cytological characteristics of recombination in birds

a Haploid genome mass for each species according to Gregory (2017). *C values for the terns and Eurasian Hobby are unknown, therefore we used C for the most closely related species Thalasseus sandvicensis and Falco peregrinus, correspondingly b Detected by staining with phosphotungstic acid in pigeon and by immunolocalization of MLH1 in the other species

Greater Rhea(Rhea americana)Domestic Goose(Anser anser)Domestic Duck(Anas platyrhynchos)F 40 1.46 278.7 61 3050 0.22 2.1 del Priore and Pigozzi (2017)F 40 1.30 283 ± 41 73.6 ± 7.8 3680 0.26 2.9 Torgasheva and Borodin (2017)M 40 1.30 281 ± 40 58.9 ± 7.6 2945 0.21 2.3 Torgasheva and Borodin (2017)F 40 1.44 – 55.9 ± 3.8 2795 – 2.0 (Pigozzi and del Priore 2016)Domestic Chicken (Gallus gallus)F 39 1.28 163 65.0 ± 4.0 3250 0.40 2.6 Pigozzi (2001)Japanese Quail(Coturnix japonica)F 39 1.35 239 ± 34 55.3 ± 2.1 2765 0.23 2.1 Calderon and Pigozzi (2006)M 39 1.35 231 ± 29 56.3 ± 1.8 2815 0.24 2.1 Calderon and Pigozzi (2006)Domestic Pigeon(Columba domestica)F 40 1.54 228 ± 22 62.7 ± 4.9 3135 0.28 2.1 Pigozzi and Solari(1999)M 40 1.54 248 ± 21 64.7 ± 4.8 3235 0.26 2.1 (Pigozzi and Solari 1999)Zebra Finch(Taeniopygia guttata)F 39 1.25 154 ± 25 45.7 ± 0.4 2285 0.30 1.9 (Calderon and Pigozzi 2006)M 39 1.25 141 ± 9 45.2 ± 0.2 2260 0.32 1.9 Calderon and Pigozzi (2006)Common Tern (Sterna hirundo)F 34 1.40* 238 ± 39 44.1 ± 5.0 2205 0.19 1.6 Lisachov et al.(2017a)Black Tern (Chlidonias niger)F 37 1.40* 288 ± 47 53.0 ± 4.2 2650 0.18 1.9 Lisachov et al.(2017a)Common Swift(Apus apus)M 39 1.38 208 ± 32 51.4 ± 4.3 2570 0.25 1.9 This paper Eurasian Hobby(Falco subbuteo)M 25 1.45* 258 ± 50 51.1 ± 6.6 2555 0.20 1.8 This paper

Karyotyping

Mitotic chromosome preparations were obtained from short-term bone marrow cell culture of the Common Swift. The culture was incubated for 2 h at 37 °C in culture medium with 10 μg/mL colchicine. Hypotonic treatment was performed with 0.56% KCl solution for 15 min at 37 °C and followed by centrifugation for 5 min at 500×g. Fresh coldfixative solution (methanol: glacial acetic acid = 3:1 v/v) was changed tree times. Cell suspension was dropped on the wet cooled slides. The slides were dried for 2 h at 65 °C and stained for 4 min with 1 μg/mL solution of DAPI in 2× SSC. The slides were then washed in deionized water, dried at room temperature and mounted in Vectashield antifade mounting medium.

Spermatocyte spreading and immunostaining

Spermatocyte spreads were prepared from testes using a drying-down technique (Peters et al. 1997). Immunostaining was performed as described by Anderson et al. (1999). Primary antibodies used in this study were as follows: rabbit polyclonal to SYCP3 (1:500; ab150292,Abcam), mouse monoclonal to MLH1 (1:50; ab14206,Abcam), and human anti-centromere (ACA) (1:100,Cat #15-235-0001, Antibodies Inc.). The secondary antibodies used were Cy3-conjugated goat anti-rabbit(1:500; Cat#111-165-144, Jackson ImmunoResearch),FITC-conjugated goat anti-mouse (1:50; Cat#115-095-003, Jackson ImmunoResearch), and AMCA-conjugated donkey anti-human (1:100, Cat#709-155-098,Jackson ImmunoResearch). All antibodies were diluted in PBT (3% bovine serum albumin, 0.1% Tween 20 in phosphate buffered saline). A solution of 10% PBT was used to perform a blocking reaction. Primary antibody incubations were performed overnight in a humid chamber at 37°, secondary antibody incubations were performed for 1 h at 37 °C. Finally, slides were mounted in Vectashield with or without DAPI (Vector Laboratories).

The preparations were visualized with an Axioplan 2 imaging microscope (Carl Zeiss) equipped with a CCD camera (CV M300, JAI), CHROMA filter sets,and the ISIS4 image-processing package (MetaSystems GmbH). Brightness and contrast of all images were enhanced using Corel PaintShop Photo Pro X6 (Corel Corp).

同时值得注意的是,在图9中,对应耦合间距d=170 nm的透射谱线中,出现了共振劈裂的现象,这是由于工艺缺陷等因素,微环侧壁出现了类似布拉格光栅的褶皱,使得微环腔中出现互耦合现象,微环腔理论上的最佳互耦合品质因数Qum与实际的互耦合品质因数Qu存在较大差异造成的[23],并不影响本文的研究结论。

Chromosome measurements and generation of recombination maps

The centromeres were identified by ACA foci. MLH1 signals were only scored if they were localized on SCs. The length of the SC of each chromosome arm was measured in micrometers and the positions of centromeres and MLH1 foci in relation to the centromere were recorded using MicroMeasure 3.3 (Reeves 2001). Individual SCs of macrochromosomes were identified by their relative lengths and centromeric indexes. To generate recombination maps of the macrochromosomes, we calculated the absolute position of each MLH1 focus multiplying the relative position of each focus by the average absolute length of the chromosome arm. These data were pooled for each arm and graphed to represent a recombination map. We measured absolute distances between two MLH1 foci within the arm in acrocentric and metacentric SCs and across the centromere in metacentric SCs.The relative distances were calculated as fractions of the SC length.

Ethics approval

Results

Karyotypes

Somatic (Fig. 1a, b) and pachytene (Figs. 2a, 3a) karyotypes of the Common Swift consist of 39 chromosome pairs (2n = 78). Macrochromosomes 1, 3 and 5 are metacentrics, 2, 4 and 6 are submetacentrics, all the other chromosomes are telocentrics forming a row gradually decreasing in length.

Fig. 1 Metaphase plate (a) and karyotype (b) of the Common Swift. DAPI-staining. Bar: 5 µm

Fig. 2 SCs of the Common Swift (a) and Eurasian Hobby (b) after immunolocalization of SYCP3 (red), MLH1 (green) and centromere proteins (blue).The arrow points to metacentric microchromosome (m). The insert is a close-up of SC1. Arrowheads point to strong (s) and weak (w) centromere signals at the SC1. Bar: 10 µm

Fig. 3 Ideogram of pachytene chromosomes of the Common Swift (a) and Eurasian Hobby (b). On the y-axis: SC length in µm. Black circles indicate centromeres

Pachytene karyotype of the Eurasian Hobby comprises 25 chromosome pairs (2n = 50). Its largest macrochromosome (SC1) and one of the microchromosomes are metacentrics, all the other chromosomes are telocentrics (Figs. 2b, 3b).

In the majority of pachytene cells (40 of 61), SC1 contained two distinct centromere signals 3.9 ± 1.6 μm away from each other. One was strong, similar to that of the other chromosome centromeres, the other was very weak. We have often observed MLH1 foci between the two centromere signals.

Apparently, one of the middle-sized macrochromosomes in both species is Z chromosome. However, it could not be identified for its size and morphology. In the Needle-tailed Swift (Hirundapus caudacutus), the species distantly related the Common Swift, Z chromosome is the smallest of metacentric macrochromosomes(Christidis 1990). In all Falco species examined it is middle-sized acrocentric chromosome (Nishida et al. 2008).

MLH1 focus number and distribution along macrochromosomes

To estimate the recombination rate and distribution of crossovers along the chromosomes of the species examined, we analyzed 1131 MLH1 foci at 858 SCs in 22 pachytene cells of the Common Swift and 3119 MLH1 foci at 1525 SCs in 61 pachytene cells of the Eurasian Hobby. The total length of SC in the swift was signi ficantly smaller than that in the hobby (208.2 ± 31.8 and 257.9 ± 49.8 μm, correspondingly, p ＜ 0.05).

The number of MLH1 foci per pachytene cell was almost the same in both species: 51.4 ± 4.3 in the swift and 51.1 ± 6.6 in the hobby, p ＞ 0.05. To estimate the total length of the recombination map in centimorgans (cM), we multiplied the average number of MLH1 foci per cell by 50 map units (one recombination event = 50 cM). The resulting estimates were 2570 cM in the swift and 2555 cM in the hobby. Recombination rate (RR) measured as the ratio of the total genetic map length in cM to the genome size in Mb was also similar in both species. However, the recombination density (RD)estimated as the number of MLH1 foci per cell per 1 μm of SC was higher in the swift (0.25) than in the hobby(0.20) (Table 1).

The patterns of MLH1 distribution along the six largest macrochromosomes reveal drastic differences between swift and hobby (Fig. 4). The swift SCs demonstrated an extremely polarized distribution of MLH1 foci. Most of them are located at the distal chromosome ends (Fig. 4a).By contrast, the hobby SCs showed rather an even distribution of MLH1 foci. The frequency of the foci was somewhat elevated at both ends of the metacentric SC1 and acrocentric SC2-SC6. Polarization was more prominent in SC6, but still less pronounced than in the metacentric SC of the swift. Suppression of the MLH1 foci around the centromere, typical for most recombination landscapes described so far, was rather weak in the metacentric SCs of both species. In all acrocentric bivalents we observed an increased MLH1 focus frequency near the centromeres.

Competing interests

随着1964年美国、加拿大两国开始实施签订的哥伦比亚河条约，大古力水电站上游的加拿大境内修建了麦卡、阿罗和邓肯3座水库，美国境内修建了利比水库，共取得有效库容315亿m3，连同大古力水库的有效库容64.5亿m3在内，有效库容共达380亿m3，再加上干支流上其它水库，总共有效库容达470.5亿m3，相当于大古力水电站坝址平均年径流量的49%，使大古力水电站成为被上游大量径流补偿的水电站，提高了其可靠出力。

与既往教育有所不同，小学数学教育信息化的提出和实施，虽然具有广阔的前景，但是在教育工作和任务的具体实践过程中，还是要按照一定的原则来实施，这样才能在问题的有效解决、综合改进过程中，不断地创造出较高的价值。首先，小学数学教育信息化，必须按照多元化的原则来进行，尤其是在动态影响因素的作用下，有针对性地干预和引导，促使小学数学教育信息化的水平获得持续性的提升。其次，在小学数学教育信息化的运作过程中，应坚持大幅度地提升教育的综合效用，促使师生沟通交流更加频繁，教师要按照求同存异的方法来教育，这对尊重小学生的主体地位，能够产生良好的效果。

Discussion

In this study, we for thefirst time describe the somatic and pachytene karyotypes of the male Common Swift and the pachytene karyotype of the Eurasian Hobby and estimate the overall number and distribution of recombination events along the chromosomes of these species.

The karyotype of the Common Swift consists of three metacentric, three submetacentric and two telocentric macrochromosomes and 31 telocentric microchromosomes (2n = 78; FN = 90). It differs from the karyotypes of the previously described related Apodidae species:Little Swift (Apus affnis affnis) (2n = ± 70) (Yadav et al. 1995), Fork-tailed Swift (Apus pacificus) (2n = 62),Needle-tailed Swift (H. caudacutus) (2n = 64) (Christidis 1990) and two tropical swift species in the genus Streptoprocne: S. biscutata (2n = 64) and S. zonaris (2n = 66)(Torres et al. 2004). Thus, the Common Swift has a karyotype that is typical of most Neoaves and apparently basal for Apodidae. The karyotype evolution of other swifts has probably involved fusions of some microchromosomes with each other or/and macrochromosomes and pericentric inversions of macrochromosomes (Torres et al. 2004). Apparently, the reduction of chromosome numbers occurred independently in the genera Apus,Hirundapus and Streptoprocne, which diverged from each other about 30–40 Mya (Jetz et al. 2012).

Fig. 4 Distribution of MLH1 foci along individual SCs in pachytene spermatocytes of the Common Swift (a) and Eurasian Hobby (b). On the x-axis:the relative position of MLH1 foci at the six largest macroSCs in relation to the centromere (black triangle). The width of the interval is 1 μm. On the y-axis: the proportion of MLH1 focus number in each interval. Colors indicate bivalents with 1–9 MLH1 foci per bivalent. The scale shows the color codes

Table 2 Recombination features of individual bivalents of the Common Swift and Eurasian Hobby

Common Swift SC1 30.1 ± 7.6 3.8 ± 1.1 0.32 ± 0.05 62 SC2 26.6 ± 6.8 3.2 ± 1.0 0.34 ± 0.04 50 SC3 21.0 ± 5.8 3.0 ± 0.9 0.43 ± 0.06 44 SC4 19.5 ± 6.4 3.0 ± 0.7 0.47 ± 0.06 41 SC5 13.4 ± 3.5 2.0 ± 0.5 0.74 ± 0.05 23 SC6 12.2 ± 2.7 2.1 ± 0.4 0.67 ± 0.03 23 Eurasian Hobby SC1 27.5 ± 6.8 4.8 ± 1.1 0.23 ± 0.01 240 SC2 27.5 ± 6.5 4.6 ± 1.2 0.22 ± 0.01 211 SC3 24.8 ± 5.1 4.2 ± 1.3 0.24 ± 0.01 188 SC4 23.3 ± 5.0 3.7 ± 0.9 0.28 ± 0.01 163 SC5 21.6 ± 4.8 3.9 ± 1.0 0.26 ± 0.02 174 SC6 19.1 ± 4.2 3.2 ± 0.9 0.32 ± 0.02 134

The karyotype of our specimen of the Eurasian Hobby coincides with those reported earlier for this species in the diploid number (2n = 50) but differs for the chromosome morphology (Christidis 1990; Nishida et al. 2008;Wang and Chen 1998). Its chromosome 1 and one of the microchromosomes are metacentrics, all the other chromosomes are telocentrics. Christidis (1990) referring to a conference report of X. Bian and Q. Li indicated that all chromosomes in the hobby karyotype were telocentric with exception of chromosome 1, which was acrocentric.Wang and Chen (1998) described the karyotypes of two hobby specimens from China. Both of them had 2n = 50,but differed in macrochromosome morphology. A male from Qiqihar city had metacentric chromosome 1, subtelocentric chromosomes 2, 4 and 5, submetacentric chromosome 9 and all the other chromosomes as telocentrics.A female from Dalian city differed from this specimen by the morphology of chromosomes 2, 4, and 5: they were telocentric. It is likely that this species is polymorphic for pericentric inversions or/and centromere shifts or/and the presence of additional heterochromatic short arms at some chromosomes.

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This work was supported by the Russian Foundation for Basic Research (Grant# 16-04-00087) and the Federal Agency for Scientific Organizations via the Institute of Cytology and Genetics (Grant # 0324-2018-0019). The funding bodies play no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript.

A rather surprising result of our study is that despite an about 40% difference in 2n and FN, the swift and the hobby have almost the same number of recombination nodules per genome. Apparently, the decrease of the lower limit of recombination rate (the number of obligatory crossovers) in the hobby has been compensated by a relaxation of the higher limit: increase in SC length and decrease in crossover interference. Similar in overall recombination rate as they are, the swift and hobby have very different patterns of recombination event distribution along macrochromosomes: extremely polarized in the swift and rather even in the hobby. A relatively even crossover distribution along the macrochromosomes with moderate peaks at the chromosome ends(including pericentromeric ends of acrocentrics) has been observed in the majority of bird species examined cytologically. A polarized distribution is more typical of mammalian chromosomes (Borodin et al. 2009; Segura et al. 2013) and macrochromosomes of anole lizards(Lisachov et al. 2017c). In birds, a pattern like this has so far been described in Zebra Finch and is considered as unique (Calderon and Pigozzi 2006; Backström et al.2010).

Conclusions

Our data add two more species to the list of birds in which the number and distribution of recombination nodules have been examined (Table 1). This list is short;however, it already reveals some important features of recombination in birds. It confirms earlier suggestions that recombination rate in birds is substantially higher than in mammals. At the same time, birds demonstrate rather a low interspecies variably of recombination rate compared to mammals. This may in part be due to a strong conservation of chromosome number in birds.However, our data demonstrate that reduction of chromosome number does not lead to decrease of recombination rate. Meanwhile, more data from different taxa are needed to draw statistically supported conclusions about the evolution of recombination in birds.

每年汛期洪水发生期，水体浑浊度高，含沙量大。农药、化肥、垃圾等各种杂物被洪水带入水体，易滋生细菌，造成有机污染与化学污染。而且雨季一般气温较高，杂物极易变质污染水体。目前水体净化完全依靠自来水厂，每到上游入洪时，自来水厂水处理便出现困难，不能满足用水需求。

Authors’ contributions

AT and PB provided the research idea and designed the experiments. LM, ES,and AT conducted the experiments and collected the data. LM and ATfinished the data analysis and compiled the results. AT and PB supervised the research and wrote the paper. All authors read and approved thefinal manuscript.

Author details

由表4可知，硅-焓方程法计算得出的热储温度为182.36 ℃～274.58 ℃，冷水混合比例为39.47%～85.88%；硅-焓图解法计算的结果为172.58 ℃～258.23 ℃，冷水混入比例为39.19%～86.46%。对比发现由混合模型计算的热储温度与Na-K温标计算的温度较为接近，与其他温标及实测情况偏离较大。

那么，琴曲是如何有意境，即如何“远”的呢？一言以蔽之，以气贯通。而此气是由生理之气、琴曲之气组成，并非单一而就的。

1 Institute of Cytology and Genetics, Russian Academy of Sciences, Siberian Department, Novosibirsk, Russia 630090. 2 Novosibirsk State University, Novosibirsk, Russia 630090. 3 Bird of Prey Rehabilitation Centre, Novosibirsk, Russia 630090.

Acknowledgements

TPACK框架是将教学法、学科以及技术进行整合，促进三者的动态平衡[5]，以呈现最优化的教学状态。徐春华等人通过问卷调查法得出高校教师TPACK处于中等能力，使用技术能力偏低，提出了提高教师TPACK能力的四点发展策略[6]。任秀华等人在分析教师知识结构的基础上，构建了高校教师TPACK知识结构框架，并提出这个结构有七个要素[7]。因此，基于TPACK框架的高校教师专业发展要求教师结合教学情境，将三者融合，呈现出最佳教学方案并实施。

We thank the Microscopic Center of the Siberian Department of the Russian Academy of Sciences for granting access to microscopic equipment.

Crossover interference plays an important role in the determination of the number and distribution of crossovers along chromosomes (Berchowitz and Copenhaver 2010; Segura et al. 2013). In this study, we measured the interference by relative distances between the neighboring MLH1 foci at the macrochromosomes: the shorter the distance, the weaker the interference. Table 2 shows that the swift chromosomes accommodated less MLH1 foci than the hobby chromosomes of comparable size.The relative distances between the foci at the swift SCs were 1.5–2-fold longer than those at the hobby SCs. This is indicative of a higher crossover interference of the swift macrochromosomes.

The authors declare that they have no competing interests.

Availability of data and materials

An adult male Common Swift with fatal accident trauma was provided by the Bird Rehabilitation Centre of Novosibirsk and euthanized in our laboratory. The testes ofan adult Eurasian Hobby were collected immediately after the bird’s death in the Centre and transported to the laboratory in cold PBS. The birds were handled and euthanized in accordance with the approved national guidelines for the care and use of laboratory animals. All experiments were approved by the Ethics Committee on Animal Care and Use of the Institute of Cytology and Genetics of Siberian Department of the Russian Academy of Sciences, Russia (approval No. 35 of October 26,2016). No additional permits are required for research on non-listed species in Russia.

Consent for publication

Not applicable.

Statistica 6.0 software package (StatSoft) was used for descriptive statistics. All results were expressed as mean ± SD; p ＜ 0.05 was considered as statistically significant.

The birds were handled and euthanized in accordance with the approved national guidelines for the care and use of laboratory animals. All experiments were approved by the Ethics Committee on Animal Care and Use of the Institute of Cytology and Genetics of Siberian Department of the Russian Academy of Sciences, Russia (approval No. 35 of October 26, 2016). No additional permits are required for research on non-listed species in Russia.

Funding

Two centromere signals that we detected at the hobby SC1 might indicate heterozygosity for pericentric inversion or a centromere shift, or the presence of vestigial inactive centromere sequence at one or both homologs. Although pericentric inversions and centromere shifts are well documented mechanisms of bird chromosome evolution (Kasai et al. 2003; Skinner and Griffn 2012; Griffn and Burt 2014), it is unlikely that they are the cause of the double centromere signal in the hobby. The occurrence of MLH1 foci between two centromere signals disapproves inversion heterozygosity,and the difference in signal intensity disapproves centromere shift. If there had been inversion heterozygosity,no crossovers would have occurred in the non-homologously synapsed inverted region. If there had been centromere shift, both signals would have been of the same intensity and weaker than the signals of the centromeres of other chromosomes. The vestigial inactive centromere sequence is the most likely explanation of the observed feature. Inactive centromeres in yeasts, plants and mammals lack several centromere proteins, such as CENP-A,CENP-C and CENP-E (Ross et al. 2015). If that were the case with the bird chromosomes, that would explain the weaker signal of the secondary centromere at the hobby SC1.

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