Heterochromatin distribution and histone modification patterns of H 4 K 5 acetylation and H 3 S 10 phosphorylation in Capsicum L .

We evaluated domesticated Capsicum pepper species from Brazil using classical and molecular cytogenetic techniques, and describe here for the first time their immunostaining patterns using anti-H4K5ac and anti-H3S10ph antibodies. All accessions showed 2n = 24 chromosomes with metacentric and submetacentric morphologies. CMA3 patterns ranged from four terminal bands in most accessions to 18 variable CMA++/DAPIand CMA+/DAPI0 bands. AntiH4K5ac signals were detected in the decondensed terminal euchromatin of most chromosome arms, indicating gene-rich open chromatin regions. Anti-H3S10ph signals were restricted to pericentromeric regions and were associated with sister chromatid cohesions and/or the condensation of all mitotic chromosomes. These karyological analyses constitute valuable diagnostic tools for identifying cultivated peppers, will be important to Capsicum breeding programs and will provide additional cytogenetic features that can be used for the conservation of capsicum genetic resources.

Capsicum is native to the tropical and subtropical Americas, with Brazil being considered a secondary center of its genetic diversity (Mongkolporn and Taylor 2011).Peppers constitute part of the Brazilian cultural and genetic heritage and are cultivated throughout that country with a variety of local names, types, shapes, colors, flavors, pungencies and aromas (Reyes-Escogido et al. 2011).Capsaicin (8-methyl-N-vanillyl-6-nonanamide) is an alkaloid exclusive to LV Martins et al. the capsicums, and their protein, vitamin and fiber contents lend them high nutritional value as well as antimicrobial, antioxidant, anti-inflammatory and anti-carcinogenic qualities (Singh and Jain 2016).
Despite their known socio-economic importance, our cytological knowledge of peppers is still incipient and further studies based on biochemical, molecular and cytogenetic techniques are needed (Moscone et al. 1993).Karyotype analysis is an important tool for characterizing Capsicum species (Moscone et al. 2007) and complements morphological, phytogeographical and molecular approaches.Cytogenetic data, associated with cytomolecular techniques and studies of karyotype evolution have aided in the correct delimitations of Capsicum -a monophyletic group with a complex taxonomy and numerous unresolved interspecific relationships (Sehr et al. 2013, Carrizo García et al. 2016).
There are two different basal chromosome numbers known for the genus: x = 12 and x = 13.Although no consensus exists yet concerning the ancestral chromosome number of that group, the presence of more symmetrical karyotypes among species showing x = 12 has led to the assumption that it is the probable ancestral number for the genus, and that the more asymmetrical karyotypes with x = 13 are derived.While 2n = 2x = 24 species have symmetrical karyotypes, low DNA amount and simple heterochromatic banding patterns, 2n = 2x = 26 species have asymmetric karyotypes and higher DNA and heterochromatin contents.Different inter-and intra-specific cytotypes are common and indicate very active processes of chromosome diversification in that group (Moscone et al. 2007, Teodoro-Pardo et al. 2007, Scaldaferro et al. 2013, Moreira et al. 2017).
The packing status of DNA associated with proteins can provide a better understanding of genetic and epigenetic regulation in eukaryotes, including vegetables (Heslop-Harrison and Schwarzacher 2011).Post-translational modifications on the N-terminal tails of histones play essential and distinct roles in chromosome organization (Marcon-Tavares et al. 2014).The acetylation of lysine 5 on histone H4 (one of the most well-studied modifications) is generally associated with actively transcribed open chromatin, and is directly involved in the control of chromatin replication (Zhang et al. 2015).The phosphorylation of serine 10 on histone H3 during mitosis and meiosis, on the other hand, seems to be associated with cell division control.The temporal and spatial distribution of H3S10ph is currently known only for few angiosperms, and immunostaining data is almost nonexistent for Capsicum.
Thus, in light of the need to provide more cytogenetic information concerning the genomic structures of peppers to aid breeding programs, we characterized accessions from the Capsicum Germplasm Active Bank at the Federal University of Piauí (BAGC-UFPI) using Giemsa staining, CMA/DAPI banding techniques and, for first time, immunostaining techniques using anti-H4K5ac and H3S10ph antibodies.Those immunostaining techniques should allow the identification and localization of modified histones associated with gene-rich regions (characterized by open chromatin; anti-H4K5ac) and regions of chromatid cohesion and/or chromosome condensation (anti-H3S10ph) to provide detailed maps of those two markers in mitotic chromosomes of Capsicum and better define pepper karyotypes.

Plant materials
Capsicum seeds were obtained from accessions held at BAGC-UFPI (Table 1).

Chromosome preparation
Root tips obtained from germinated seeds were pretreated with p-dichlorobenzene (0.015 g mL -1 ) for 2 hours at room temperature and then fixed in Carnoy (ethanol: acetic acid v/v) for at least 12 hours at -20 °C.

Conventional staining
Slide preparation followed the methodology described by Guerra (2002).Root tips were hydrolyzed in 5N HCl for 20 minutes at room temperature and then squashed in 45% acetic acid.The slides were subsequently frozen in liquid nitrogen, stained with Giemsa for 10-15 min and mounted with Entellan (Merck).

Immunostaining with H4K5ac and H3S10ph antibodies
The immunostaining technique followed the protocol of Feitoza and Guerra (2011a).Roots tips fixed in 4% paraformaldehyde were washed in 1 × PBS and incubated overnight at 4 °C with the primary anti-H4K5ac and anti-H3S10ph antibodies (rabbit polyclonal IgG-Upstate Biotechnology, USA) diluted 1:300 in 1× PBS containing 3% BSA.To detect the primary antibodies, 15 µL of secondary antibody FITC-conjugated goat anti-rabbit IgG (Sigma) diluted 1:60 in 3% BSA was added, and the root tips held in a dark chamber at room temperature for 3 hours.The preparations were then mounted in DAPI (2 mg mL -1 ): Vectashield (1:1 v/v) and immediately photographed.

Image analyses and morphometry
The best five metaphases of each accession were photographed using a DFC345Fx digital camera coupled to a Leica DM2500 microscope.The images were adjusted and optimized for brightness and contrast using Adobe Photoshop CS3.Chromosome sizes were determined using the Micromeasure 3.3 program supplemented by Microsoft Excel 2010 software.Idiograms were constructed using Corel Draw X7 and chromosome morphologies were classified according to Guerra (1988).

RESULTS AND DISCUSSION
Conventional staining allowed a detailed description of the interphase nuclei, prophase condensation patterns, chromosome numbers, as well as chromosome morphologies and sizes (Figure 1, Table 1).Giemsa staining revealed semi-reticulate nuclei (Figure 1a) and Solanum-type prophase condensation pattern (CP) with early condensed proximal regions and late condensing terminal chromatin (Feitoza Costa and Guerra 2017).All accessions showed 2n = 24 chromosomes, with metacentric and submetacentric morphologies.As reported by Moscone et al. (2007), all domesticated Capsicum taxa have a chromosome base number x = 12 with symmetrical karyotypes, while other semi-domesticated and several wild species (such as C. campylopodium, C. villousum and C. buforum) have x = 13 with asymmetrical karyotypes that are probably derived from centric fissions (Robertsonian).annuum accessions from the Capsicum germplasm bank in Mexico showed high TCL polymorphisms, demonstrating the high inter-and intraspecific chromosome diversity found in Capsicum (Teodoro-Pardo et al. 2007).The observed differences in chromosome sizes of the different accessions could be related to unequal degrees of chromosome contraction during cell division (Moscone 1990), differences in pretreatment and chromosome condensation (Pozzobon et al. 2006), and/ or to different classes of repetitive DNA sequences as evolutionary components of pepper genome structures based on constitutive heterochromatin expansions (Shcherban 2015, Scaldaferro et al. 2016).
GC-rich heterochromatin is universal in Capsicum, appearing in all taxa with x = 12; although located mainly in terminal regions, it may also occur in variable numbers of small intercalary and distal bands that are difficult to detect (as in C. chacoense, C. annuum var.glabriusculum, C. recurvatum, C. villosum and C. flexuosum) (Scaldaferro et al. 2013).Heterochromatin distribution is not always homogeneous within or among species, and polymorphisms have been reported (reviewed by Guerra 2000).Moscone et al. (1993) observed centromeric, distal, and intercalary bands unequally distributed in both chromosome arms of peppers.Those authors classified domesticated species into two groups based on their C-band contents: the white-flowered group (C.annuum, C. chinense, and C. frutescens) with low heterochromatin contents, small terminal bands and lacking intercalary bands; and the purple-flowered group (which includes C. campylopodium and C. pubescens) with high heterochromatin contents, large telomeric bands and intercalary blocks.Although C. baccatum belongs to the white-flowered subgroup, it differs from the other three domesticated species by showing a complex heterochromatin pattern and longer chromosome lengths.We note here that C. baccatum var.pendulum exhibits higher numbers of heteromorphic GC-rich bands as compared to C. annuum, C. chinense, and C. frutescens.
For a better understanding of chromatin structural patterns and cell cycles in Capsicum, we performed immunostaining using antibodies against histones modified by the acetylation of lysine 5 of histone H4 (H4K5ac) and the phosphorylation of serine 10 of histone H3 (H3S10ph).
Anti-H4K5 patterns were classified as Solanum -type (Feitoza Costa and Guerra 2017), with labeling restricted to diffuse chromatin of the nuclei and in the decondensed terminal euchromatin of most mitotic chromosomes, from prophase-metaphase (Figure 4); the chromocenters of the interphase nuclei (and some terminal heterochromatic regions) were not stained.The acetylation patterns we observed showed labeling tightly associated with terminal open chromatin in the mitotic chromosomes of C. baccatum var.pendulum and C. chinense.Similar labeling has been reported in other plants with small chromosomes and wellestablished condensation pattern, such as Costus spiralis, Eleutherine bulbosa (Feitoza and Guerra 2011b) and P. vulgaris (Fonseca et al. 2014).The fact that anti-H4K5ac markings were present in all of the cell cycle phases analyzed suggests their close relationships with chromatin dynamic states that may change during the cell cycle and become restricted to specific chromosome sites during meiosis (Feitoza and Guerra 2011b).
Additionally, hyperacetylated regions (immunostaining marking) coincided with late condensing euchromatic regions (conventional staining), while hypoacetylated or nonacetylated regions were directly linked to early condensing prophase chromatin and to CMA ++ or CMA + blocks (CMA/ DAPI staining).As suggested by Dhar et al. (2009), acetylation patterns are inversely related to heterochromatic patterns: late replicating euchromatin (including heterochromatin) is early condensing, whereas early replicating chromatin (gene-rich euchromatin) is late condensing.Since H4K5 acetylation is universally associated with gene expression and open chromatin (as observed in tomatoes [Cigliano et al. 2013], Silene and Allium cepa [Chang et al. 2008]), we believe that its labelling in uncondensed euchromatin is related to potentially active regions in the chromosomes of Capsicum.
Anti-H3S10ph signals, on the other hand, were restricted to the pericentromeric regions of all of the chromosomes during prometaphase-metaphase, while interphase nuclei were not stained (Figure 5).This pattern is associated with mitotic condensation and/or chromatid cohesion regardless of eu-and heterochromatin regions involved (unlike the pattern observed for anti-H4K5 markings).That phosphorylation marking only occurred in pericentromeric regions of the A chromosomes in Cestrum strigilatum (another Solanaceae species), although an exception was observed in its B chromosomes (Fernandes et al. 2008).
These results, associated with previous work performed in other socio-economically important crops, reinforce the idea that the hyperacetylation of H4K5 can be used as a chromosome marker for detecting active euchromatin associated with gene expression.H3S10 phosphorylation seems to be involved in regulating sister chromatid cohesion, although further studies will be needed.Our results demonstrated the feasibility of using immunostaining to detect gene-rich and pericentromeric regions during the cell cycle of Capsicum that could be used in future studies involving cytomolecular mapping and contribute to the genetic improvement of peppers and their preservation in Brazil.

Figure 1 .
Figure 1.Chromosome number of prometaphase and metaphase in Capsicum sp. by Giemsa staining.a-interphase nucleus; a'prometaphase with arrows indicating chromosomic pair with decondensed chromatin in terminal region; b, c -prometaphase; c', d, e and f -metaphase.Arrows in c', e and f indicate NORs.Bar = 10 µm.

Figure 3 .
Figure 3. Idiograms representing size, morphology and distribution of CMA ++ terminals bands (larger yellow bands) and CMA + bands (smaller yellow bands) on chromosome of Capsicum's accessions.

ACKNOWLEDGMENTS
We would like to thank the Brazilian agency CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico/process number 457201/2014-2) for financial support.