FISH-mapping of the 5 S rDNA locus in chili peppers ( Capsicum-Solanaceae )

We present here the physical mapping of the 5S rDNA locus in six wild and fi ve cultivated taxa of Capsicum by means of a genus-specifi c FISH probe. In all taxa, a single 5S locus per haploid genome that persistently mapped onto the short arm of a unique metacentric chromosome pair at intercalar position, was found. 5S FISH signals of almost the same size and brightness intensity were observed in all the analyzed taxa. This is the fi rst cytological characterization of the 5S in wild taxa of Capsicum by using a genus-derived probe, and the most exhaustive and comprehensive in the chili peppers up to now. The information provided here will aid the cytomolecular characterization of pepper germplasm to evaluate variability and can be instrumental to integrate physical, genetic and genomic maps already generated in the genus.


INTRODUCTION
Capsicum L., or chili peppers, (Solanaceae) is a New Word genus distributed from Mexico to Argentina that comprises 31 species, several of which are of considerable economic importance.Five Capsicum species (C.annuum L. var.annuum, C. chinense Jacq., C. frutescens L., C. baccatum L. var.pendulum (Willd.)Eshbaugh and var.umbilicatum (Vellozo) Hunz.et Barboza, and C. pubescens Ruiz et Pav.) were domesticated by American natives and are now cultivated as part of the human diet (Moscone et al. 2007).In fact, after tomato, peppers are the most culivated fruit vegetable species in the world (FAOSTAT 2014).
In plants, 5S rRNA genes are organized in one or several loci, arranged as tandem repeats of thousand to hundreds of thousand copies per genome, with each copy comprising a coding region (CR; ~120 bp) and a non-transcribed intergenic spacer (NTS; ~100-900 bp) (Cloix et al. 2001 and references therein).
Previous fluorescent in situ hybridization (FISH) assays revealed the loci number and localization of the 5S rRNA gene in only a few Capsicum species, by means of different probes, PATRICIA M. AGUILERA et al. some of them derived from unrelated taxa (Park et al. 1999, 2000, Scaldaferro et al. 2006, Kwon and Kim 2009).Similarly, other economically important members of the Solanaceae family such as tomato, tobacco and petunia have been studied to reveal the presence of 5S sites (Garcia et al. 2012) but information on potato and eggplant is still absent.In addition, more comprehensive approaches led to the development of several pepper maps (Wu et al. 2009 and references therein) and recently, to the genome sequencing of wild and cultivated species of chili peppers (Kim et al. 2014, Qin et al. 2014).However, despite 45S rRNA genes have been mapped onto a pepper genetic linkage map (Wu et al. 2009), information regarding 5S rDNA genes in Capsicum, is still lacking.
The main purpose of this contribution is to achieve a physical mapping of the 5S rDNA locus in wild and cultivated taxa of Capsicum by means of a genus-specifi c FISH probe.The cytological 5S rDNA marker is highly useful in the identifi cation of individual chromosomes for further germplasm characterization.In addition, the information reported here, not only broadly expands the limited knowledge of 5S rDNA mapping in Capsicum, but will also be undoubtedly useful to integrate physical, genetic and genomic maps already generated in the genus (Tanksley et al. 1988, Kang et al. 1997, Wu et al. 2009, Qin et al. 2014, Kim et al. 2014).

PLANT MATERIAL
Five cultivated in addition to six wild taxa of Capsicum were employed to cytologically map the 5S rRNA gene.Taxa, provenances, herbarium specimens and status of the studied material are summarized in Table I.Plant material was determined by Dr. Gloria E. Barboza.

CHROMOSOME PREPARATIONS
Chromosomes of somatic metaphases were observed in root tip squashes obtained from seed germination.Seeds were placed on moisturized fi lter paper in Petri dishes and germinated at room temperature (about 25 ºC).Roots were collected when they reached 2 cm in length and were immediately pretreated with p-dichlorobenzene saturated solution for 3 h at room temperature and then fi xed in Farmer solution (absolute ethanol : glacial acetic acid, 3 : 1) for a minimum of 12 h at 4 ºC.Somatic chromosome spreads were prepared following the procedure described by Schwarzacher et al. (1980).Root tips were macerated in 2% (m/v) cellulase (Onozuka R-10 from Trichoderma viridae; Serva, Heidelberg, Germany) plus 10% (v/v) pectinase (from Aspergillus niger; Sigma, St. Louis, Missouri, USA), dissolved in 40% glycerol in 0.01 mol/L citric acid/sodium citrate buffer, pH 4.8, at 37 ºC for 2 h, and then squashed in 45% acetic acid.After removal of the coverslip with CO 2 , slides were air dried, incubated for 1-2 days at room temperature, and then kept at 4 ºC until use.

DNA ISOLATION AND 5S PROBE PREPARATION
Genomic DNA was isolated and purifi ed from fresh leaves of C. pubescens by using the CTAB method, with minor modifications (Rogers and Bendich 1994).An additional step of DNA purification using phenol-chloroform and a RNAse A treatment (Invitrogen, USA) was added, followed by DNA precipitation with ethanol (Sambrook et al. 1989).Sample quality was checked by electrophoresis in agarose 0.8% gel, and by measurements of Abs.260 nm/280 nm index, to confirm DNA integrity and absence of RNA contamination.The 5S rDNA unit of C. pubescens was amplifi ed through polymerase chain reaction (PCR) by using two oligonucleotide primers, P1 5'-GATCCCATCAGAACTCC-3' (17mer) and P2 5'-  from Park et al. (2000).Both primer sequences bind to the highly conserved sections in the coding region of the 5S gene (Fig. 1).The PCR reaction mixture comprised 10 ng of template DNA from  C. pubescens, 0.5 pmol of each primer, 200 mM of each dNTP, 5 μl of 10X reaction buffer and 1 unit of Taq DNA polymerase "Sequencing Grade" (Promega, USA).Thirty six amplifi cation cycles were performed, each involving denaturation at 94 °C for 1 min, primer annealing at 57 °C for 1 min, and extension at 72 °C for 1 min.Amplifi ed 5S rDNA fragments were separated in agarose 1.4% gel and then were excised from the gel and purifi ed by using the GFX kit (Amersham Pharmacia, USA).Amplifi ed fragments were cloned into the pCR2.1-TOPOvector (Invitrogen, USA) and transformed in Escherichia coli "TOP10 One Shot" (Invitrogen, USA), following the manufacturer`s instructions.The obtained clones were grown in LB liquid selective medium.Plasmidic DNA was isolated through mini-preparations, digested with restriction enzymes and visualized in agarose 1% gel to check the insert stability.The nucleotide sequences of the clones were determined (Macrogen, Korea) and Cp5S-3 was characterized (Fig. 1) and selected for further FISH analysis.Intercalary markers were mapped using the index di = d x 100/a (d = distance of band center from the centromere; a = length of the corresponding chromosome arm) according to Greilhuber and Speta (1976).

GENUS SPECIFIC PROBE DEVELOPMENT AND CHARACTERIZATION
The generation and molecular characterization of the 5S rDNA probe from C. pubescens is displayed in Figure 1a-d.Clone Cp5S-3 is 297 bp in length and exhibits, at the sequence level, all the specifi c features of functional 5S rDNA units.

FISH EXPERIMENTS
A Capsicum-derived rDNA probe (Cp5S-3) was used in FISH experiments in order to cytologically map the 5S rDNA locus in the chili peppers.
The somatic chromosome number added to the karyotype formula and the number and position of the 5S rDNA loci per diploid complement in the analyzed taxa are summarized in Table I.Additional features associated to the chromosome that carries the 5S rDNA are also provided in Table I, i.e. the chromosome length (CL), the centromeric index (CI) and the relative chromosome length (RCL).
All the studied taxa are diploid and have 2n = 24, except for C. rhomboideum (Dunal) Kuntze which has 2n = 26 chromosomes.Karyotype formula varied among taxa but symmetry is a core feature in Capsicum.

LOCI NUMBER, SIZE AND DISTRIBUTION
FISH experiments revealed a unique chromosome pair hybridized with the Cp5S-3 probe (green dots) in the eleven examined taxa (Fig. 2a).
Polymorphisms for these loci within individuals or between taxa were not observed.Therefore, a single 5S locus per haploid genome was found in Capsicum.5S FISH signals of almost the same size and brightness intensity were observed in the studied taxa of Capsicum (Fig. 2a, b).In the analyzed taxa, the 5S rDNA locus persistently mapped onto the short arm of a unique metacentric chromosome pair, at intercalar position (Fig. 2a I and Fig. 2b).

FISH PROBE CHARACTERIZATION
According to genic and non transcribed spacer (NTS) motives, the FISH probe derived clone Cp5S-3 is fairly representative of functional 5S segments.Gene features, i.e. the internal control region (ICR) formed by the motives Box A, Box C and IE of Cloix et al. (2001) and the RNA binding domains (RNA BD) to the TFIIIA (Kellogg and Appels 1995), were identifi ed in the primary sequence and are represented in Figure 1d.Further structures in the NTS, i.e. the transcription termination site (TTS) and the TATA-like box (-29), the GC dinucleotides (-13) and the C (-1) at 3`end region, are also associated to functionality (Cloix et al. 2001).
FISH EXPERIMENTS Cytological mapping of the 5S has been previously performed in several chili peppers.Scaldaferro et al. (2006) used a probe derived from Beta vulgaris (pXV1) on wild and cultivated taxa.Furthermore, Park et al. (1999Park et al. ( , 2000) ) used a wheat derived-probe (pScT7) and a Capsicum-related one, respectively, on cultivated taxa, the same as Kwon and Kim (2009).However, ours is the first report on the cytological characterization of the 5S in wild taxa of Capsicum by using a genus-specifi c FISH probe.Moreover, our results in C. eximium, C. fl exuosum, C. rhomboideum and both wild and cultivated varieties of C. baccatum (praetermissum and pendulum) are novel in the genus.Furthermore, considering the number of analyzed taxa, our contribution is the most exhaustive and extensive characterization of the 5S locus in chili peppers.

LOCI NUMBER AND DISTRIBUTION
Our results on the FISH location in the wild and cultivated taxa of Capsicum revealed a single 5S locus per haploid genome.This locus is regularly mapped onto the short arm of a unique metacentric chromosome pair of approximately the same size among taxa, with the exception of the CL of C. fl exuosum and C. rhomboideum.All the analyzed taxa exhibited the 5S locus at an intercalar position, which is closer to the distal portion of the short   et al. 1999, 2000, Scaldaferro et al. 2006, Kwon and Kim 2009).Altogether, the combined results evidence the universality of this cytological feature in Capsicum.Physical location of the 5S in the same chromosome pair regarding our accessions and those of Scaldaferro et al. (2006), was also found in C. cardenasii (#9), C. pubescens (#3) and cultivated varieties of C. baccatum (#5).In contrast to the latter authors who found the 5S loci at the chromosome pair #6 of C. annuum var.annuum, we detected it at #3.According to Park et al. (2000), the 5S loci in the cultivated taxa of Capsicum was assigned to chromosome 1 by the synteny relationship with the corresponding linkage group of tomato, of Lapitan et al. (1991).Furthermore, Kwon and Kim (2009) assigned the 5S loci of C. annuum to the linkage group 7 of the genetic map of Kang et al. (1997).However, the 5S loci was not mapped in the recent contributions of the genome of chili peppers (Qin et al. 2014, Kim et al. 2014).

CONCLUSIONS
The main purpose of this contribution was to achieve a physical mapping of the 5S rDNA locus in wild and cultivated taxa of Capsicum by means of a genus-specifi c FISH probe.This cytological marker is useful to identify individual chromosomes in order to assist the cytomolecular characterization of pepper germplasm by the evaluation of inter and PATRICIA M. AGUILERA et al.
intraspecifi c cytogenetic variability.Additionally, the information reported here can be used to integrate physical, genetic and genomic maps already generated in the genus which undoubtedly will represent a valuable tool for breeding purposes of chili peppers.

Figure 1 -
Figure 1 -Development and molecular characterization of the 5S rDNA probe from C. pubescens.(a-b) Gel electrophoresis of the PCR amplifi cation products and clones, respectively, with that related to the FISH probe marked with arrowheads; (c) Amplifi cation strategy according to the 5S rDNA unit organization and primers P1/P2 from Park et al. (2000); (d) Core features of the FISH probe Cp5S-3; in the gene, the internal control region (ICR) formed by the motives Box A, Box C and IE (Cloix et al. 2001) and the RNA binding domains (RNA BD) to the TFIIIA (Kellogg and Appels 1995) are shown; additional features correspond to the non transcribed spacer (NTS); TTS, transcription termination site.

Figure 2 -
Figure 2 -Physical mapping of the 5S rDNA locus in the wild and cultivated Capsicum taxa by means of FISH.(a) Well-arranged karyograms derived from DAPI (blue) stained somatic metaphase plates showing the number and position of the 5S rDNA locus (green dots) added to further elementary cytological features; chromosomes other than metacentric (m) are solely marked (sm, st); (b) Schematic depiction of the chromosomes that carry the 5S rDNA locus considering the mean values described in Table I; green dots size relates to the mean fl uorescence intensity.(See the colors in the on-line version).