Centromeres of Cucumis melo L. comprise Cmcent and two novel repeats, CmSat162 and CmSat189.

Centromeres are prerequisite for accurate segregation and are landmarks of primary constrictions of metaphase chromosomes in eukaryotes. In melon, high-copy-number satellite DNAs (SatDNAs) were found at various chromosomal locations such as centromeric, pericentromeric, and subtelomeric regions. In the present study, utilizing the published draft genome sequence of melon, two new SatDNAs (CmSat162 and CmSat189) of melon were identified and their chromosomal distributions were confirmed using fluorescence in situ hybridization. DNA probes prepared from these SatDNAs were successfully hybridized to melon somatic and meiotic chromosomes. CmSat162 was located on 12 pairs of melon chromosomes and co-localized with the centromeric repeat, Cmcent, at the centromeric regions. In contrast, CmSat189 was found to be located not only on centromeric regions but also on specific regions of the chromosomes, allowing the characterization of individual chromosomes of melon. It was also shown that these SatDNAs were transcribed in melon. These results suggest that CmSat162 and CmSat189 might have some functions at the centromeric regions.

Introduction

Repetitive DNA sequences form a major portion of nuclear DNA in eukaryotic genomes, particularly in melon, accounting for 42% of the total sequence [1,2]. Repetitive DNA sequences are organism-specific at the species or genus level and/or chromosomal location-specific including centromeric or subtelomeric [3]. Repetitive DNAs are classified into two major groups, namely, tandem repeats (micro-, mini-, or satellite DNA) and dispersed repeats, i.e., transposable elements (DNA transposons and retrotransposons). Tandem repeats are arranged in tandem arrays of monomeric units [4], whereas dispersed repeats are mobile and are scattered across the genome [5,6]. Satellite DNA (SatDNA) families are in a special class of tandemly repeated monomers in heterochromatic regions comprising 150–400 base pairs (bp) of DNA [3,4,7].

Melon (Cucumis melo L.) belongs to Cucurbitaceae family and is a diploid species possessing 2n = 2x = 24 chromosomes [8]. The relatively large amount (20%-30%) of SatDNAs in Cucurbitaceae serves as an interesting resource for the identification of new SatDNA [9]. pSat107 is a melon-specific SatDNA with a nucleotide sequence length of 352 bp [10] and it hybridizes to melon centromeres [11,12,13]. Centromeres are important for sister chromatid segregation during cell division. Heterochromatic regions are characterized as those with accumulation of SatDNAs and favorable sites for centromeres [14]. Plant centromeres are composed of satellite DNA repeats and highly repeated centromere-specific retrotransposons [15]. Functional centromeres are determined by the occurrence of nucleosomes containing centromere-specific histone H3 (CENH3), the binding of which to DNA can be analyzed by chromatin immunoprecipitation [16,17,18,19,20].

The melon genome is 454 mega-base pairs (Mb) in size [1]. To date, only Cmcent has been reported as a centromere marker in melon [12], and there are no reports on other centromeric repeats in melon. Utilizing the draft melon genome sequence, we identified two new SatDNAs, namely, CmSat162 and CmSat189, as melon centromeric repeats. Here we describe successful hybridization of them to melon somatic chromosomes and pachytene chromosomes, and their distribution on the chromosomes which can be used for chromosome identification. We also discuss transcription of these SatDNA repeats to identify their possible functions in centromere structures.

Materials and methods

Plant materials

Three Indonesian melon cultivars,‘Baladewa’ (Cucumis melo L. subsp. melo var. cantalupo Ser.), ‘Ivory F1 hybrids’ (C. melo L. subsp. melo var. inodorus) and ‘P90’ (C. melo L. subsp. agrestis var. conomon), were used in this study. The seeds were germinated on moistened filter paper in petri dishes and grown in a growth chamber at 25°C.

Data mining and dot plot identification of SatDNA repeats

Scaffold sequences of Cucumis melo DHL92 (BioProject accession PRJEB68; [21]) were retrieved from the National Center for Biotechnology Information (NCBI) database and subjected to tandem repeat sequence analysis using Tandem Repeat Finder version 4.09 (http://tandem.bu.edu/trf/trf.basic.submit.html). New SatDNA sequences were identified in the melon genomic scaffold sequence “LN681816” (S1 Table). Five tandem repeats were detected, and two of them (CmSat162 and CmSat189) were used. The consensus sequences of these SatDNAs and their sequence alignments are shown in S2 Table, S1 and S2 Figs. The selected sequences were blasted against the melon genome database (http://cucurbitgenomics.org/blast), and the primers were designed using FastPCR software [22].

Genomic DNA isolation

Genomic DNA was extracted using a modified version of the method of Doyle and Doyle [23]. In brief, 0.5 g of young leaves were ground in 500 μL of cetyltrimethylammonium bromide (CTAB) isolation buffer [2% (w/v) CTAB, 1.4 M NaCl, 20 mM EDTA, 100 mM Tris-HCl pH. 8.0, 0.2% (v/v) 2-mercaptoethanol] that had previously been incubated at 60°C in a preheated mortar before transferring into 1.5-ml tubes. The samples were incubated at 60°C for 30 min in a water bath with occasional gentle swirling. Next, 200 μL of 24:1 (v/v) chloroform:isoamyl alcohol (CIAA) was added and mixed gently but thoroughly, and the mixture was centrifuged at 1600 x g for 15 min at RT followed by removal of the aqueous phase at the top of the tube. Finally, the samples were transferred into a new 1.5-ml tube, and the genomic DNA was purified using CIAA twice. Then, 1/10 volume of 3 M sodium acetate and 2/3 volume of cold isopropanol were consecutively added. The samples were kept at -30°C for 1 h to precipitate the DNA and centrifuged at 500 x g for 2 min. The supernatant was poured off, followed by the addition of 600 μL of DNA wash buffer (60 mM potassium acetate, 10 mM Tris-HCl pH 7.5, 60% ethanol). Subsequently, the tubes were centrifuged at 10,000 x g for 1 min, and then the supernatant was poured off again. The samples were washed twice with DNA wash buffer and air-dried using the Automatic Environmental SpeedVac System AES1010 for 30 min. The pellet was re-suspended in 100 μL of TE buffer (10 mM Tris-HCl pH 7.4, 1 mM EDTA). One μL of RNase A (Qiagen) at 1 mg/μL was added and incubated at 37°C for 1 h. Finally, the DNA quality was analyzed by electrophoresis.

Total RNA isolation

Total RNA was extracted from leaves by a slightly modified version of a method described elsewhere (http://www.patentsencyclopedia.com/app/20090111114). In brief, 0.5 g of each sample was ground into powder in liquid nitrogen in a chilled mortar and pestle with 300 μL of RNA isolation buffer [48% (w/v) guanidine thiocyanate (GTC), 10 mM 2-morpholinoethanesulfonic acid (MES) pH 6.46, 1% (w/v) polyvinylpyrrolidone (PVP), and 0.2% (v/v) 2-mercaptoethanol]. The mixture was transferred into 2-ml tubes to which 500 μL of CIAA was added and mixed gently. The samples were centrifuged at 10,000 x g for 15 min. The aqueous phase was transferred into a PD column, centrifuged at 10,000 x g for 1 min, and the supernatant was discarded. Six hundred μL of 40% diethylene glycol dimethyl ether (Diglyme) was added to the tube before centrifugation at 10,000 x g for 1 min. The supernatant was carefully discarded from the tube to avoid removing the loosely attached pellet. Next, 600 μL of 80% ethanol was added to the tube to purify the pellet, followed by centrifugation at 10,000 x g for 1 min. The washing steps with Diglyme and ethanol were repeated twice. The PD column was centrifuged at 10,000 x g for 1 min to completely dry the column. Finally, 30 μL of pre-warmed sterile distilled water was added and centrifuged at 10,000 x g for 1 min. The total RNA was treated with Deoxyribonuclease RT Grade (Nippon Gene, Japan) to remove the genomic DNA in accordance with the manufacturer’s instructions.

Cloning of satellite DNA repeats

All SatDNA repeats used are listed in Table 1. The monomer length of CmSat162 and CmSat189 (162 bp and 189 bp, respectively) were determined based on bioinformatic analysis. These repeats were isolated by polymerase chain reaction (PCR) amplification of ‘P90’ genomic DNA using the oligonucleotide primer pairs 5’-GGATTGTCGTACTTGAACACTTGGT-3’ and 5’-CCTAAGTAGTGTTCATGAGGTGCCT-3’, and 5’- CACATCATAACAAGTGTATCAACA-3’ and 5’-TCATCCACGAAGCATGATAC-3’, respectively. The resulting 311-bp and 267-bp PCR products amplified from the dimers were cloned into pGEM-T-Easy Vector (Promega) in accordance with the manufacturer’s protocol.

Table 1

SatDNA repeats used in this study.

Name	Type	Length (bp)	Source
CmSat162	Satellite DNA	162*	This study
CmSat189	Satellite DNA	189*	This study
Cmcent	Satellite DNA	354	Koo et al. 2010

*: The monomer length of these SatDNAs were identified using Tandem Repeat Finder from melon scaffold sequence (acc. No. LN681816) deposited in NCBI GenBank.

Semi-quantitative PCR amplification of CmSat162 and CmSat189 transcripts

First-strand cDNAs were synthesized from 0.5 μg of total RNA using ReverTraAce® qPCR RT Master Mix with gDNA Remover (Toyobo, Japan). The resulting cDNA was used as a template in a 30-μl PCR reaction volume using gene-specific primers of CmSat162 and CmSat189. Semi-quantitative PCR (sqPCR) was performed with a PCR Thermal Cycler Dice™ Touch (Takara, Japan) using TaKaRa Ex Taq Hot Start Version (TaKaRa, Japan). The sqPCR products were separated on 2% agarose gel and stained with ethidium bromide before visualization using High Performance UV Transluminator (USA). The β-actin gene was used as an internal control for determining the sqPCR amplification efficiency in the tissue samples, and it was amplified using the primer pair PbActin2f1 and PbActin2r1 [24].

Chromosome and probe preparations and fluorescence in situ hybridization (FISH)

The preparations of mitotic metaphase and meiotic pachytene chromosomes were conducted using modified Carnoy’s solution II in accordance with the work of Setiawan et al. [13]. The Cmcent probe was labeled with Biotin-Nick translation mix (Roche), whereas CmSat162 and CmSat189 were labeled with Dig-Nick translation mix (Roche). The FISH protocol as described by Setiawan et al. [13] was followed. For the pachytene chromosomes, the hybridization mixtures were added on the chromosome preparations, covered with a 22 x 40-mm cover slip and sealed with rubber cement. The slides were denatured on a hot plate at 80°C for 2–3 min. Finally, the slides were placed in a humidity chamber and incubated at 37°C overnight. Detection solutions of 126 μL [1% BSA in 4x SSC 125 μl + 0.4 μl/ml anti-digoxigenin rhodamine (Roche) 0.5 μL + 0.5 μg/mL biotinylated streptavidin-FITC (Vector Laboratories) 0.5 μl] were used and washed in 2x and 0.1x SSC for 3 min after incubation at 37°C for 30 min. Finally, the slides were counterstained with 4,6-diamidino-2-phenylindole (DAPI) in a VectaShield antifade solution (Vector Laboratories).

Sequence comparison and image analysis

The comparison among Cmcent, CmSat162, and CmSat189 sequences was performed using a dot plot and analyzed using Unipro UGENE software. Karyotyping ideograms were constructed using CHIAS IV [25]. FISH signals were observed under a fluorescence microscope (Olympus BX53) equipped with a cooled CCD camera (Photometrics CoolSNAP MYO), processed using Metamorph, Metavue imaging series version 7.8, and edited using Adobe Photoshop CS 6.

Results

CmSat162, a major component of melon centromeres

We conducted physical localization of CmSat162 and Cmcent on somatic metaphase chromosomes, and meiotic pachytene chromosomes. CmSat162 produced major FISH signals on all melon centromeres, and it co-localized with Cmcent, a previously known melon centromere marker (Fig 1). pSat107 and Cmcent shared high sequence homology (Fig 3d), and pSat107 was identified to be Cmcent [20]. CmSat162 and Cmcent were successfully hybridized on metaphase cells of ‘Baladewa’, ‘Ivory F1 hybrids’, and ‘P90’ (Fig 1d, 1h and 1l). High-resolution FISH on ‘P90’ pachytene chromosomes was carried out to reveal the organization of these SatDNAs (Fig 1m–1p). Both repeats were located within a heterochromatic block of primary constrictions (Fig 1p). These results suggest that CmSat162 is an additional new centromeric marker in melon beside Cmcent.

Fig 1

Physical mapping of SatDNAs on melon mitotic and meiotic chromosomes, and extended DNA fibers using FISH.

Metaphase chromosomes of ‘Baladewa’ (a-d)), ‘Ivory F1 hybrids’ (e-h), ‘P90’ (i-l), and pachytene chromosome of ‘P90’ (m-p). Cmcent (green) and CmSat162 (red). Scale bars = 10 μm.

Fig 3

Sequence comparison analysis between Cmcent and CmSat162 (a), Cmcent and CmSat189 (b), CmSat162 and CmSat189 (c), and Cmcent and pSat107 (d) using dot plot, and expression analysis of CmSat162 and CmSat189 amplified from leaf tissues of melon (f). The positions of monomers and PCR primers of CmSat162 and CmSat189 are indicated with black horizontal bars and arrows above the dot plot, respectively. Monomer of Cmcent was used for the dot plot analysis. Ideogram of melon using prometaphase chromosomes based on condensation patterns, and locations of Cmcent and CmSat189 hybridization signals (e). Centromere signals (yellow) resulted from co-localization of signals of Cmcent and CmSat189, CmSat189 signals (red), heterochromatic region (black), and euchromatic region (gray).

CmSat189, a part of the melon centromeres

CmSat189 was successfully hybridized to all chromosomes of ‘Ivory F1 hybrids’ (Fig 2). This probe produced major signals on primary constrictions and additional ones on chromosome-specific regions, permitting characterization of individual homologous chromosomes (Fig 2c and 2d). The majority of the signals co-localized with those of Cmcent and CmSat162, although they shared low sequence homologies (Figs 2a, 2b and 3a–3c, and S3 Fig). Some of them were located at interstitial, pericentromeric, or subtelomeric regions depending on the chromosome pairs (Fig 2d, Table 2); e.g., chromosome 1 was distinguished from others with the presence of the signals of CmSat189 at the centromeric and interstitial regions of the short arms with strong intensities, at the interstitial region of the long arms with medium intensities, and at the pericentromeric and subtelomeric regions of the long arms with weak intensities (Fig 2d). An ideogram was constructed using prometaphase chromosomes based on the condensation patterns of heterochromatic and euchromatic regions, as well as Cmcent and CmSat189 hybridization signals (Fig 3e).

Fig 2

Physical mapping of CmSat189 on melon mitotic chromosomes of ‘Ivory F1 hybrids’.

FISH detection of Cmcent (a) and CmSat189 (b) in prometaphase chromosomes. Overlay image of Cmcent and CmSat189 (c). Homologous chromosomes are arranged from left to right in the order of their lengths (No. 1 to No. 12), and based on the locations of CmSat189 and Cmcent signals (d). Cmcent (green) and CmSat189 (red). Scale bars = 10 μm.

Table 2

Features of melon chromosomes based on CmSat189 hybridization signals.

Chromosome number	Chromosome features based on CmSat189 hybridization signals
1	Highest signal intensity at centromeric region, high at interstitial region of 1S, medium at interstitial region of 1L, and weak at pericentromeric and subtelomeric regions of 1L.
2	Highest signal intensity at centromeric region and weak at interstitial region of 2L.
3	Highest signal intensity at centromeric region, medium at interstitial region, and weak at pericentromeric and subtelomeric regions of 3L.
4	Highest signal intensity at centromeric region and weak at pericentromeric region and interstitial regions of 4L.
5	High signal intensity at centromeric region, medium at interstitial regions, and weak at pericentromeric and subtelomeric regions of 5L.
6	Highest signal intensity at centromeric region, medium at interstitial region, and weak at pericentromeric region of 6L.
7	High signal intensity at centromeric region, medium at interstitial region, and weak at pericentromeric and subtelomeric regions of 7L.
8	Highest signal intensity at centromeric region, high at pericentromeric region, and weak at interstitial region of 8L.
9	High signal intensity at centromeric region and medium at interstitial region of 9L.
10	Highest signal intensity at centromeric region, weak at interstitial region of 10S, and medium at interstitial and subtelomeric regions of 10L.
11	Highest signal intensity at centromeric region, medium at pericentromeric and interstitial regions, and weak at subtelomeric regions of 11L.
12	Highest signal intensity at centromeric region and medium at pericentromeric and interstitial regions of 12L.

Transcription of CmSat162 and CmSat189 in melon

The expression of CmSat162 and CmSat189 was examined using semi-quantitative PCR with repeat-specific primers (Fig 3f). The result showed that both repeats were expressed in leaf tissue of melon, and the expression level was similar to that of the endogenous control, the β-actin gene. This suggests that both SatDNAs are actively transcribed in melon with some specific unknown functions.

Discussion

Repetitive sequences, particularly SatDNAs, are found with high copy numbers within eukaryotic genomes. They are primarily located at centromeric, pericentromeric, and telomeric regions, which form the major components of heterochromatin [26]. They play important roles in biological processes related to cellular and chromosomal functions [3,27]. Therefore, understanding the organization and function of SatDNAs should contribute toward accelerating molecular cytogenetic research in higher plants. Furthermore, SatDNAs can be used to identify homologous chromosomes and determine the positions of centromeres in plants [11,12,14,20,28]. Utilizing the melon genome sequence, we identified new SatDNAs and detected their distributions in melon chromosomes.

Centromeres play essential roles in sister chromatid cohesion, and they are predominantly composed of SatDNAs and retrotransposons [15]. In the present study, five tandem repeats were detected from the published DNA sequence LN681816, and two tandem repeats (CmSat162 and CmSat189) were used (S1 Table). The monomer length of CmSat162 (162 bp) is similar to that of the centromeric SatDNAs of Type III in cucumber [14], pAL1 in Arabidopsis [29], pTS5 in Beta procumbens [30](Gindullis et al. 2001), and CentO in Oryza [31]. In this study, we conducted physical mapping of Cmcent and CmSat162 on mitotic and meiotic chromosomes. CmSat162 was located precisely at primary constrictions and flanked by the Cmcent signals, which are known to be located in centromeric regions. Although CmSat162 and Cmcent share similar centromeric locations, the sequences of these two repeats are completely different (Fig 3a and S2 Table). These results indicate that CmSat162 is a new centromeric repetitive sequence, and melon centromeres are composed not only of Cmcent but also of CmSat162.

CmSat189 produced chromosome-specific signals not only at centromeric regions that were flanked by Cmcent but also at pericentromeric, interstitial, and subtelomeric regions. Using this probe, we elucidated its position and distribution in melon chromosomes. CmSat162 and CmSat189, both of them were exclusively hybridized on the primary constrictions of melon. In addition, CmSat162, CmSat189, and Cmcent repeats shared low sequence homology (Fig 3a–3c), and their organizations shared high homology with Cucumis melo genomic scaffold sequence (accession number: LN681816) (S3 Fig). Thus, our findings suggest that CmSat162 and CmSat189 are new repetitive sequences for melon centromeres.

Although the centromere plays an important role in chromosome segregation during the cell cycle, its function is conserved and regulated by epigenetic mechanisms [32]. The expression of centromeric SatDNAs has been reported in plant species such as maize [33], Arabidopsis thaliana [34], rice [35,36], and banana [37]. The transcription of centromeric repetitive sequences, particularly SatDNAs, has essential functions not only in heterochromatin formation but also in maintaining centromere structures [26]. Bouzinba-segard et al. [38] reported minor satellite transcripts of 120 bp in murine cells that localize to centromeres. Forced accumulation of 120 bp transcripts leads to defects in chromosome segregation and sister-chromatid cohesion, changes in hallmark centromeric epigenetic markers, and mislocalization of centromere-associated proteins essential for centromere function. Moreover, Rošić et al. [39] reported that the 359-bp satellite III (SAT III) of D. melanogaster was localized in centromeric regions of all major chromosomes and produced a long noncoding RNA. Depletion of SAT III RNA led to mitotic defects not only of the sex chromosome but also of all autosomes. SAT III RNA binds to the kinetochore component CENP-C and is required for correct localization of the centromere-defining proteins CENP-A and CENP-C, as well as outer kinetochore proteins. Thus, it is suggested that centromeric RNAs maybe play important roles in centromere function. In this study, CmSat162 and CmSat189 were shown to be actively transcribed in melon. These SatDNAs are maybe involved in the maintenance of melon centromere stability similar to those reported in maize by Gent et al. [40], where the authors revealed that diverse DNA sequences and multiple types of genetic elements in and near maize centromeres support centromere functions and constrain centromere positions. However, further analysis of the contributions of CmSat162 and CmSat189 to centromere stability is required to elucidate the specific functions of these two SatDNAs. Currently, there are no functional data of centromeric DNA repeats in Cucumis species, particularly in melon, that can be identified by chromatin immunoprecipitation (ChIP) using anti-CENH3 antibody followed by sequencing of the immunoprecipitated DNA (ChIP-Seq). The identification of melon centromeric region is solely conducted by FISH. Therefore, the ChIP-Seq is necessary to be conducted in the future to reveal functional centromeric repeats in melon.

The consensus sequence of CmSat162 and is sequence alignment analyzed by Tandem Repeat Finder.

(DOCX)

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The consensus sequences of Cmsat189 and its sequence alignment analyzed by Tandem Repeat Finder.

(DOCX)

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CmSat162 (A) and CmSat189 (B) repeat organization in Cucumis melo genomic scaffold sequence (accession number: LN681816).

For Cmsat162, sequence from nt1141769 to nt1144288 of LN681816 was used for dot plot analysis whereas sequence from nt1875724 to nt1877241 was used for CmSat189. The consensus sequences of both CmSat162 and CmSat189 are listed in S2 Table.

(DOCX)

Click here for additional data file.

SatDNA repeats on melon DNA sequence “LN681816” analyzed by Tandem Repeat Finder ver. 4.09.

(PDF)

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The consensus sequences of CmSat162 and CmSat189 acquired from LN681816 DNA sequence analyzed by Tandem Repeat Finder ver. 4.09.

(DOCX)

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(JPG)

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4 Oct 2019

PONE-D-19-26046

Centromeres of Cucumis melo L. comprises Cmcent and two novel repeats, CmSat162 and CmSat189

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Reviewer #1: The manuscript “Centromeres of Cucumis melo L. comprises Cmcent and two novel repeats, CmSat162 and CmSat189” by Setiawan and others describes the identification of two new tandem repeat sequences. These two sequences largely colocalize with the established centromeric sequence Cmcent at the primary constriction. Melon centromere evolution is particularly interesting as species of this genus show evolutionary centromere repositioning events. These events are common in animals, but not in plants. What the role of centromeric sequences are in centromere repositioning events are remains unknown. Expanding our knowledge of what types of sequences are associated with centromeres, and whether these sequences are limited to the centromere or also found along the chromosome arms is important for understanding the evolutionary dynamic behavior of these fast evolving sequences. This work provides novel insights in satellite DNA in the very interesting Cucumis genus. Several concerns remain and are listed below.

+ The authors used scaffolds to identify new tandem repeats sequences using TRF. Could the authors provide the TRF settings used? Also, could the authors provide a reasoning why they chose to use scaffolds to identify novel tandem repeats, rather than WGS data? WGS data allows you to assemble from sequences that not only present in the scaffolds. Melters et al 2013 Genome Biology used this very approach to identify hundreds of candidate centromere tandem repeats. This approach would also provide the authors with an estimated quantity of the sequence identified.

+ Were the CmSat162 and CmSat189 sequences found the genome of other Cucumis species? A BLASTn search would give a quick indication.

+ What were the sizes of the FISH probes? In our hands, monomer sized FISH probes allow for very specific hybridization, whereas longer probes might hybridize with less specificity.

+ At the end of the introduction, the authors introduce the histone variant CENH3. For clarity, please specify that nucleosomes containing CENH3 instead of H3 mark the functional centromere.

+ In the final paragraph of the introduction, it is unclear what question the authors set out to answer in this manuscript.

+ Figure 1: did the authors also check for CmSat162 signal on pachytene chromosomes from ‘Baladewa’ and ‘Ivory F1 hybrids’? Ideally, this is reported as well.

+ Figure 2: did the authors also check for CmSat189 signal on ‘Baladewa’ and ‘P90’ chromosomes? Especially the karyotype ideogram is very informative. Showing this for all three cultivars and for both novel tandem repeats would provide a great resource for the field.

+ Do CmSat162 and CmSat189 colocalize on (pachytene) chromosomes of the three tested cultivars?

+ It is very interesting that transcripts of CmSat162 and CmSat189 were found by sqPCR. Were these transcripts cell cycle specific? Can this be determined from leave tissue? In addition, the -RT control, ideally a control with and without RNase, and with and without DNase is done to guarantee that no DNA contamination persisted.

Reviewer #2: This paper describes two satellites from melon that the authors show are cytologically located at the centromeres, and other locations. They also show that these satellite sequences are transcribed.

The presentation of the satellite sequences is somewhat confusing. The authors identify the 162 bp and 189 bp satellites using Tandem Repeat finder on a genomic scaffold. According to Table S1, the 162 bp repeat family and the 189 bp repeat family show 76% and 90% identity, respectively. They describe cloning the repeats using primers that amplify dimers of the repeats, but the primers given in the methods only partially match the satellite monomer sequences given in Table S2, making it unclear what sequences were actually cloned, and whether the monomers in Table S2 are particular examples of monomers or are consensus sequences. In Figure 3, comparing dimers of the two satellites to each other and to the previously identified satellite Cmcent, there is limited symmetry between the two halves of the dimers, especially for the 162 bp satellite, suggesting considerable divergence in the sequences of the two monomers used in the display. The authors also fail to mention how long Cmcent is and whether a monomer or dimer is displayed. They do mention in the Introduction that Sat107 is 352 bp long and is centromeric, but they do not identify this with Cmcent. There needs to be a better explanation of how the primers, monomers, and dimers relate to each other. The authors should state in the text in addition to Table S1 the degree of conservation of the two satellite families, and whether the sequences in Table S2 are consensus sequences or not. It might be useful to have a supplementary figure showing the relationship of the primers, monomer sequence in Table S2, and the dimer sequences in Figure 3 to the sequences amplified from the genomic scaffold.

Do the authors know whether one or both strands of the satellites are transcribed? Were the primers used for sqPCR the same as those used for gene amplification?

It would be helpful at some point for the authors to mention that there are currently no functional data or CENH3 ChIP data to identify the centromere sequences in Cucumis, and the identification is solely by FISH.

Minor points

The title is ungrammatical (Centromeres…comprises).

Line 3: Not all centromeres are associated with repetitive sequences. Though this is common in plants and animals, it is not

true of many single-celled eukaryotes.

Line 16: I think the authors mean “portion”, not “proportion”.

In Figures 2d and 3d, it would be helpful to the reader to number the chromosomes 1-12 at the bottom of the figures.

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Reviewer #1: Yes: Daniël P Melters

Reviewer #2: No

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10 Dec 2019

We appreciate the comments of the editor and reviewers to the previous manuscript.

Submitted filename: Response to reviewers.docx

Click here for additional data file.

23 Dec 2019

Centromeres of Cucumis melo L. comprise Cmcent and two novel repeats, CmSat162 and CmSat189

PONE-D-19-26046R1

Dear Dr. Koba,

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Reviewer #1: All comments have been addressed

Reviewer #2: (No Response)

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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6. Review Comments to the Author

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Reviewer #1: This reviewer acknowledges that all raised concerns have been commented on by the authors and adequately addressed most of the concerns raised. At the same time, this reviewer had hoped that the authors would be a little more forthcoming and elaborate more on some of the concerns raised or incorporate insightful information in the main text where feasible.

+ It would be helpful to the reader if the authors mentioned in the methods section that the default settings were used. Ideally in the response, the authors could mention why they think that the default setting was the appropriate setting to use.

+ The finding that CmSat162 and CmSat189 was only found in Cucumis melo and not in other Cucumis species is noteworthy of mentioning in the main text, as this might point to a recently evolved tandem repeat, or that the other Cucumis genomes are too incomplete to draw this conclusion. Using WGS data to identify novel tandem repeat sequences might be one approach to more confidently make such a statement.

+ The authors mention that Ivory F1 hybrids chromosome were not tested for CmSat162 and Baladewa and P90 chromosomes for CmSat189. It would be insightful if the authors could elaborate why they didn’t do this and why they think this is not important to do.

+ On the question about co-localization of CmSat162 and CmSat189, could the authors elaborate why they didn’t do this experiment and why they think it would add to their story?

+ Although not important for the story, this reviewer think it would be interesting to learn what they authors think about possible cell cycle specificity (if there is any) of the transcription of CmSat162 and CmSat189.

Reviewer #2: The authors have responded to most of the comments, but there are a few places where they still need to clarify what they have done. Reviewer 1 asked for TRF settings, which the authors provided in their reply but not in the Methods. They just need to add "with default parameters" to the mention of TRF in the Methods. Likewise they should state explicity that the same primers were used for genomic cloning and for sqPCR, and that the FISH probes are the 311bp and 327bp genomic clones. For example, on p. 6 they could say "using the same gene-specific primers of CmSat162 and CmSat189 used in cloning" and on p. 7 they could say that "the cloned PCR products for CmSat162 ad CmSat189 were labeled with Dig-Nick".

The primers described do not match the consensus sequences for CmSat162 and CmSat189 shown in S2 Table and appear to have been designed from the scaffold LN681816 (1142313-1142623 and 1876253-1876496). This should be mentioned where the primers are described. These primers give products of 311bp and 327bp, which are derived from dimers of divergent monomers, not from identical consensus monomers, which would yield products of ~145bp and ~55bp. The positions of the 311bp and 327bp clones could be added to S1 fig and S2 Fig, added to S2 Table, and/or given as accession numbers in a public database.

The authors leave the impression in their reply that Figures 3a and 3b display identical tandem dimers of the 162 and 189 consensus sequences ("we make a dimer of the consensus sequence"), but this does not seem possible, or else the left and right halves of these figures would be identical, which they clearly are not. Are the dimers in these figures taken from LN681816? Please clarify, and if so provide the specific coordinates for the dimers. Also the arrows showing where the primers are located would predict the 145bp and 55bp PCR products from monomers, not the 311bp and 327bp products that they actually amplified. It would be helpful to indicate where the primers actually bind, not where they would be expected to bind if the monomers were identical.

These clarifications can be easily fixed without further outside review, but they should be fixed, so that others can build on what the authors have done.

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Reviewer #1: Yes: Daniël P. Melters

Reviewer #2: Yes: Paul B. Talbert

30 Dec 2019

PONE-D-19-26046R1

Centromeres of Cucumis melo L. comprise Cmcent and two novel repeats, CmSat162 and CmSat189

Dear Dr. Koba:

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