Grabiele, Mauro; Aguilera, Patricia Mabel; Ducasse, Daniel Adrián; Debat, Humberto Julio

Mauro Grabiele ⁷7Author for correspondence: maurograbiele@conicet.gov.ar

Universidad Nacional de Misiones, Instituto de Biología Subtropical (UNaM-CONICET) and Instituto de Biotecnología Misiones, Posadas, Misiones, Argentina.

https://orcid.org/0000-0001-7386-4924

Patricia Mabel Aguilera

Universidad Nacional de Misiones, Instituto de Biología Subtropical (UNaM-CONICET) and Instituto de Biotecnología Misiones, Posadas, Misiones, Argentina.

https://orcid.org/0000-0002-5063-9350

Daniel Adrián Ducasse

Instituto de Patología Vegetal, Centro de Investigaciones Agropecuarias (INTA), Córdoba, Argentina.

† Deceased 05-11-2020

https://orcid.org/0000-0001-6487-5007

Humberto Julio Debat

Instituto de Patología Vegetal, Centro de Investigaciones Agropecuarias (INTA), Córdoba, Argentina.

https://orcid.org/0000-0003-3056-3739

7Author for correspondence: maurograbiele@conicet.gov.ar

Area Editor: Dra. Cassia Sakuragui

†
Deceased 05-11-2020

Figuras (10)
Tablas (2)

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Figure 1
a. Amplification strategy of the 5S NTS in Capsicum via P1/P2 and RT1/RT2 primer pairs; TFIIIA BS = TFIIIA transcript binding site (Kellogg & Appels 1995Kellogg EA & Appels R (1995) Intraspecific and interspecific variation in 5S RNA genes are decoupled in diploid wheat relatives. Genetics 140: 325-343.); Box A, Box C and IE = internal control regions (Cloix et al. 2002Cloix C, Tutois S, Yukawa Y, Mathieu O, Cuvillie C, Espagnol M-E, Picard G & Tourmente S (2002) Analysis of the 5S RNA Pool in Arabidopsis thaliana: RNAs are heterogeneous and only two of the genomic 5S loci produce mature RNA. Genome Research 12: 132-144. DOI: 10.1101/gr.181301
https://doi.org/10.1101/gr.181301... ). b. PCR products of 5S regions; (P1/P2) 1 = C. flexuosum; 2 = C. praetermissum; 3 = C. baccatum var. umbilicatum; 4 = C. baccatum var. baccatum; 5 = C. annuum var. annuum; 6 = C. annuum var. glabriusculum; 7 = C. frutescens; 8 = C. chinense; 9 = C. chacoense; 10 = C. tovarii; (RT1/RT2) 11 = C. hunzikerianum; 12 = C. schottianum; 13 = C. campylopodium; 14 = C. coccineum; note the typical amplification ladder pattern of the repetitive 5S region; arrowhead point out to purified products; M, marker (Kbp); (-), negative control. c. Consensus and LOGO 5S gene sequences in Capsicum. d. Secondary structure of the consensus 5S gene in Capsicum displaying the usual eukaryote mode (Szymanski et al. 2003Szymanski M, Barciszewska MZ, Erdmann VA & Barciszewski J (2003) 5S rRNA: structure and interactions. Biochemical Journal 371: 641-651. DOI: 10.1042/BJ20020872
https://doi.org/10.1042/BJ20020872... ); red to blue colours scale correspond to high to low probabilities, respectively.

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Figure 2
Structural characterization of the 5S NTS in chili peppers. Multiple alignment and annotation of 126 sequences linked to 23 taxa of Capsicum and L. rantonnei. SR = structural regions. Functional features related to transcription of upstream (polyT) and downstream (-30 TATA, -12 GC, -1 C/T) 5S CR are shown. 1-3: L. rantonnei (Sister group); 4-8: C. rhomboideum; 9-12: C. geminifolium; 13-16: C. pereirae; 17-21: C. villosum; 22-25: C. friburgense; 26-33: C. schottianum; 34-37: C. recurvatum; 38-43: C. campylopodium; 44-51: C. hunzikerianum; 52-56: C. flexuosum; 57-62: C. coccineum; 63-68: C. pubescens; 70-73: C. eximium; 74-80: C. tovarii; 81-84: C. chacoense; 85-90: C. praetermissum; 91-93: C. baccatum var. baccatum; 94-97: C. baccatum var. umbilicatum; 98-101: C. baccatum var. pendulum; 103-107: C. frutescens; 109-113: C. chinense; 115-119: C. annuum var. glabriusculum; 120-125: C. annuum var. annuum. 69, 102, 108, 114 and 126 correspond to cultivated taxa under AF217950-4 (Park et al. 2000Park YK, Park KC, Park CH & Kim NS (2000) Chromosomal localization and sequence variation of 5S rRNA gene in five Capsicum species. Molecules and Cells 10: 18-24.). Major insertions at C. chacoense, C. tovarii and Andean clade taxa added to sites over 90% gaps are hided. Note main structural features defining clades at Capsicum.

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Figure 3
Structural characterization of the 5S NTS in chili peppers. Major insertion segments as useful markers at C. tovarii (123 nt), Andean clade taxa (13 nt) and C. chacoense (7 nt). Note the unusually large SRI of C. tovarii that contains a repeated region at 5’ and 3’ (light green) associated to a region fairly similar to a 5S gene (light red), probably originated by unequal crossing over within the ribosomal region.

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Figure 4
a-b. Probable scenario on the origin of the current SRI – a. of Atlantic Forest clade members; b. that shared by Tovarii, Baccatum and Annuum clades taxa. 1: simplified depiction of usual homologous chromosomes pairing at the ribosomal region. 2: same region misaligned by repeated sequences suffering an unequal exchange (orange triangles) with loss of segments (Del). 3: resultant SRI.

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Figure 5
Structural characterization of the 5S NTS in Solanaceae. Note the consensus sequences for each taxa and their comparable annotated functional features related to transcription of upstream (poly(T)) and downstream (-30 TATA-like, -12 GC, -1 C/T) 5S gene added to the NTS organization in three structural regions (SRI-III) according to differences on the GC content and inner variability. For details see Grabiele et al. (2020)Grabiele M, Aguilera PM, Ducasse DA & Debat HJ (2020) Data for: Molecular characterization of the 5S rDNA non transcribed spacer and reconstruction of phylogenetic relationships in Capsicum. Mendeley Data. V1. Available at <http://dx.doi.org/10.17632/xcrmb7m7y8.1>. Access on 10 Sep. 2020. DOI: 10.17632/xcrmb7m7y8.1
http://dx.doi.org/10.17632/xcrmb7m7y8.1... , Suppl. Figs. 7-10.

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Figure 6
Bootstrap phylogenetic network of the 5S NTS sequences of Capsicum and comparable related taxa -Lycianthes, Solanum, Datura, Atropa-. The network was constructed via SplitsTree4 employing the NTS alignment matrix of Grabiele et al. (2020)Grabiele M, Aguilera PM, Ducasse DA & Debat HJ (2020) Data for: Molecular characterization of the 5S rDNA non transcribed spacer and reconstruction of phylogenetic relationships in Capsicum. Mendeley Data. V1. Available at <http://dx.doi.org/10.17632/xcrmb7m7y8.1>. Access on 10 Sep. 2020. DOI: 10.17632/xcrmb7m7y8.1
http://dx.doi.org/10.17632/xcrmb7m7y8.1... , Suppl. Fig. 12, the Neighbornet algorithm, the uncorrected P distance and 100 boostrap replicates. Note the major lineages comprising Capsicum. * = C. schottianum paralogs; ** = C. villosum paralogs. Scale bar indicates the scale of the network branches.

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Figure 7
ML phylogenetic tree of the 5S NTS sequences of Capsicum and comparable related taxa -Lycianthes, Solanum, Datura, Atropa-. Phylogenetic relationships were inferred via FastTree 2.1.5 employing the NTS alignment matrix of Grabiele et al. (2020)Grabiele M, Aguilera PM, Ducasse DA & Debat HJ (2020) Data for: Molecular characterization of the 5S rDNA non transcribed spacer and reconstruction of phylogenetic relationships in Capsicum. Mendeley Data. V1. Available at <http://dx.doi.org/10.17632/xcrmb7m7y8.1>. Access on 10 Sep. 2020. DOI: 10.17632/xcrmb7m7y8.1
http://dx.doi.org/10.17632/xcrmb7m7y8.1... , Suppl. Fig. 12, the GTR+G model and 1,000 resamples. Selected support values are shown side to main branches and those below 50% are condensed. For a detailed support values depiction see Grabiele et al. (2020)Grabiele M, Aguilera PM, Ducasse DA & Debat HJ (2020) Data for: Molecular characterization of the 5S rDNA non transcribed spacer and reconstruction of phylogenetic relationships in Capsicum. Mendeley Data. V1. Available at <http://dx.doi.org/10.17632/xcrmb7m7y8.1>. Access on 10 Sep. 2020. DOI: 10.17632/xcrmb7m7y8.1
http://dx.doi.org/10.17632/xcrmb7m7y8.1... , Suppl. Fig. 13. Note the grouping of the NTS sequences at expected clades in Capsicum.

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Figure 8
a-b. Bootstrap phylogenetic network of the 5S NTS sequences of domesticated and their related wild varieties of Capsicum, and of Atlantic Forest clade members- a. of domesticated and their related wild varieties of Capsicum; b. of Atlantic Forest clade members. Networks were constructed via SplitsTree4 employing the NTS alignment matrices of Grabiele et al. (2020)Grabiele M, Aguilera PM, Ducasse DA & Debat HJ (2020) Data for: Molecular characterization of the 5S rDNA non transcribed spacer and reconstruction of phylogenetic relationships in Capsicum. Mendeley Data. V1. Available at <http://dx.doi.org/10.17632/xcrmb7m7y8.1>. Access on 10 Sep. 2020. DOI: 10.17632/xcrmb7m7y8.1
http://dx.doi.org/10.17632/xcrmb7m7y8.1... , Suppl. Fig. 1 (for a.) and Grabiele et al. (2020)Grabiele M, Aguilera PM, Ducasse DA & Debat HJ (2020) Data for: Molecular characterization of the 5S rDNA non transcribed spacer and reconstruction of phylogenetic relationships in Capsicum. Mendeley Data. V1. Available at <http://dx.doi.org/10.17632/xcrmb7m7y8.1>. Access on 10 Sep. 2020. DOI: 10.17632/xcrmb7m7y8.1
http://dx.doi.org/10.17632/xcrmb7m7y8.1... , Suppl. Fig. 2 (for b.), the Neighbornet algorithm, the uncorrected P distance and 100 boostrap replicates. pub and pubAF217954 = C. pubescens; bac = C. baccatum var. baccatum; umb and bacAF217951 = C. baccatum var. umbilicatum; pen = C. baccatum var. pendulum; fru and fruAF217952 = C. frutescens; chi and chiAF217953 = C. chinense; gla = C. annuum var. glabriusculum; ann and annAF217950 = C. annuum var. annuum; per = C. pereirae; vil = C. villosum; fri = C. friburgense; sch = C. schottianum; rec = C. recurvatum; cam = C. campylopodium; hun = C. hunzikerianum. Scale bar indicates the scale of the network branches.

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Figure 9
Main evolutionary features at the 5S NTS of Capsicum onto the ML phylogenetic tree. a = 3’ end SRI: novel CT block; middle SRII: novel CGGG motif. b = Middle SRII: major insertion (13 nt). c = 5’ end SRI: major deletion (21 nt) not affecting polyT stretch; CT block CTCTCC-type. d = SRII: almost entire deletion (C. coccineum); CT block MCTCTC-type. e = Middle SRII: partial deletion; CT-block CGTTCTGT-type. f = Near 5’ end SRII: TT to two purines. g = SRII: major block deletions, CGGG to CGGGG and two purines insertion at 3’ end; SRIII: GAT to GAC at 3’ end; CT block CCTCTT-type. h = Middle SRI: AATTT to GATTT, CT insertion, GCG to GTG; Near 5’ end SRII: C to G. i = SRI: deletion of TGTCG and insertion of a large polyT stretch (16 nt group parsimony) upstream ancestral polyT stretch (CCTTTTT). j = SRI: major insertion (123 nt) of a 5S related stretch near 3’ end. k = Middle SRIII: AGA to GGA. l = SRI: polyT stretch insertion of 16 to 21 nt (group parsimony). m = SRI: polyT stretch insertion of 16 to 12 nt (group parsimony); T to A at 3’ end of polyT stretch. n = SRI: insertion (7 nt) of repetitive nature at polyT stretch (C. chacoense). o = Near 5’ end SRII: major purine-rich block deletion (12 nt; C. praetermissum).

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Figure 10
Nucleotide substitution rates at the 5S NTS of Capsicum and comparable related taxa -Lycianthes, Solanum, Datura, Atropa-. Nucleotide diversity (π) of phylogenetically relevant groups was estimated in MEGA 7.0.26 employing the NTS alignment matrix of Grabiele et al. (2020)Grabiele M, Aguilera PM, Ducasse DA & Debat HJ (2020) Data for: Molecular characterization of the 5S rDNA non transcribed spacer and reconstruction of phylogenetic relationships in Capsicum. Mendeley Data. V1. Available at <http://dx.doi.org/10.17632/xcrmb7m7y8.1>. Access on 10 Sep. 2020. DOI: 10.17632/xcrmb7m7y8.1
http://dx.doi.org/10.17632/xcrmb7m7y8.1... , Suppl. Fig. 12, the K2+G model and 100 bootstrap replicates. Evolutionary divergence times in Solanaceae are considered (Sarkinen et al. 2013Sarkinen T, Bohs L, Olmstead RG & Knapp S (2013) A phylogenetic framework for evolutionary study of the nightshades (Solanaceae): a dated 1000-tip tree. BMC Evolutionary Biology 13: 214. DOI: 10.1186/1471-2148-13-214
https://doi.org/10.1186/1471-2148-13-214... ). SR = substitution rate; s.e. = standard error; MYA = million years ago; ROT = rest of taxa; ROC = rest of clades; CA = common ancestor. Vertical bars correspond to s.e. of SR.

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Table 1
Summary of the material of Capsicum and Lycianthes used in this study in addition to the amplification strategy and public accession of the 5S rDNA sequences.

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Table 2
Summary of main features at the NTS in Capsicum and Lycianthes.

Clade	Taxon	Provenance	Voucher specimen and herbarium	Status	Fruit colour, shape and pungency	2n	PCR primers	DDBJ/ENA/GenBank
-	Lycianthes rantonnei (Carriere) Bitter	Argentina, Córdoba	A. Romanutti 54. CORD	Wild	Red, spherical, non-pungent	24	P1-P2	MK638982-MK638984
Andean	Capsicum rhomboideum (Dunal) Kuntze	Venezuela, Táchira	Y Sánchez García 20. CORD	Wild	Red, spherical, non-pungent	26	P1-P2	MK650985- MK650989
Andean	Capsicum geminifolium (Dammer) Hunz.	Perú, Cajamarca	Leiva 3938. CORD	Wild	Red, spherical, non-pungent	26	P1-P2	MK650953- MK650956
Atlantic Forest	Capsicum pereirae Barboza et Bianchetti	Brazil, São Paulo	GE Barboza et al. 1651. CORD	Wild	Yellowish-green, spherical, pungent	26	P1-P2	MK650965- MK650968
	Capsicum villosum Sendtn.	Brazil, Rio de Janeiro	GE Barboza et al. 1653. CORD	Wild	Yellowish-green, spherical, pungent	26	P1-P2	MK651005- MK651009
	Capsicum friburgense Hunz.	Brazil, Rio de Janeiro	GE Barboza et al. 2048. CORD	Wild	Yellowish-green, spherical depressed, pungent	26	P1-P2	MK650944- MK650947
	Capsicum schottianum Sendtn.	Brazil, São Paulo	GE Barboza et al. 1650. CORD	Wild	Yellowish-green, spherical, pungent	26	RT1-RT2	MK650990- MK650997
	Capsicum recurvatum Witas	Brazil, Paraná	GE Barboza et al. 915. CORD	Wild	Yellowish-green, spherical, pungent	26	P1-P2	MK650981- MK650984
	Capsicum campylopodium Sendtn.	Brazil, Rio de Janeiro	GE Barboza et al. 2057. CORD	Wild	Yellowish-green, spherical compressed, pungent	26	RT1-RT2	MK650914- MK650919
	Capsicum hunzikerianum Barboza et Bianchetti	Brazil, São Paulo	GE Barboza et al. 1648. CORD	Wild	Yellowish-green, spherical, pungent	26	RT1-RT2	MK650957- MK650964
Flexuosum	Capsicum flexuosum Sendtn.	Argentina, Misiones	GE Barboza et al. 1034. CORD	Wild	Red, spherical depressed, variable pungent	24	P1-P2	MK650939- MK650943
Bolivian	Capsicum coccineum (Rusby) Hunz.	Bolivia, Santa Cruz	GE Barboza et al. 1916. CORD	Wild	Red, spherical, pungent	-	RT1-RT2	MK650929- MK650934
Pubescens	Capsicum pubescens Ruiz et Pav. cv. locoto rojo	Argentina, Salta	EA Moscone 256. CORD	Cult.	Red, turban-shaped, pungent	24	P1-P2	MK650975- MK650980
Purple Corolla	Capsicum eximium Hunz.	Argentina, Salta	EA Moscone 255. CORD	Wild	Red, spherical, pungent	24	P1-P2	MK650935- MK650938
Tovarii	Capsicum tovarii Eshbaugh, Smith et Nickrent	Peru, Junín	AT Hunziker & GE Barboza 25653. CORD	Wild	Red, spherical, pungent	24	P1-P2	MK650998- MK651004
Baccatum	Capsicum chacoense Hunz.	Bolivia, Tarija	GE Barboza et al. 1793. CORD	Wild	Red, spherical, pungent	24	P1-P2	MK650920- MK650923
	Capsicum praetermissum Heiser et Smith	Brazil, São Paulo	GE Barboza et al. 1646. CORD	Wild	Red, spherical-elliptic, pungent	24	P1-P2	MK650969- MK650974
	Capsicum baccatum L. var. baccatum	Bolivia, Santa Cruz	JG Seijo et al. 3812. CORD	Wild	Red, ovoid-elliptic, pungent	24	P1-P2	MK650903- MK650905
	Capsicum baccatum L. var. umbilicatum (Vellozo) Hunz. et Barboza	Argentina, Córdoba	G Bertone wo/#. CORD	Cult.	Red, umbonate-umbilicate, pungent	24	P1-P2	MK650910- MK650913
	Capsicum baccatum L. var. pendulum (Willd.) Eshbaugh cv. cayenne	Argentina, Salta	EA Moscone et al. 211. CORD	Cult.	Red, fusiform, pungent	24	P1-P2	MK650906- MK650909
Annuum	Capsicum frutescens L.	Brazil, Minas Gerais	GE Barboza 795. CORD	Cult.	Red, elongate, pungent	24	P1-P2	MK650948- MK650952
	Capsicum chinense Jacq. cv. pimenta de cheiro vermelha	Brazil, Pará	GE Barboza 807. CORD	Cult.	Red, conical, pungent	24	P1-P2	MK650924- MK650928
	Capsicum annuum L. var. glabriusculum (Dunal) Heiser et Pickersgill	USA, Texas	NMSU ID10983. CORD	Wild	Red, spherical, pungent	24	P1-P2	MK650898- MK650902
	Capsicum annuum L. var. annuum	Mexico, Sonora	NMSU ID10272. CORD	Cult.	Red, pequin, pungent	24	P1-P2	MK650892-MK650897

Clade	Taxon	#	SRI			SRII			SRIII			Full NTS
Clade	Taxon	#	PI	GC	ML	PI	GC	ML	PI	GC	ML	PI	GC	ML
-	Lycianthes rantonnei	3	96.6	37.7	58.3	99.3	69.1	95	93.3	32.2	30.0	97.1	53.1	183.3
-	Capsicum	123	52.0	35.0	68.8	50.3	70.3	76.2	81.6	35.1	29.9	55.1	50.4	174.9
Andean	Capsicum rhomboideum	5	88.9	20.7	53.2	77.6	49.5	120.8	77.7	30.7	28.0	80.3	39.3	202.0
Andean	Capsicum geminifolium	4	91.0	24.1	56.0	98.3	67.4	120.3	91.7	30.3	29.8	94.7	50.2	206.0
Atlantic Forest	Capsicum pereirae	4	89.5	38.1	44.0	75.9	74.3	96.3	96.8	33.3	30.8	81.9	57.6	171.0
	Capsicum villosum	5	90.7	36.7	41.4	78.7	71.7	101.2	88.0	30.0	30.0	82.9	56.1	172.6
	Capsicum friburgense	4	96.4	40.4	41.5	97.8	72.6	89.5	100	36.7	30.0	97.8	57.6	161.0
	Capsicum schottianum	8	93.7	35.7	40.3	78.0	73.3	93.1	94.2	32.5	30.0	84.3	56.5	163.4
	Capsicum recurvatum	4	97.6	36.6	41.0	94.3	77.6	69.3	100	33.3	30.0	96.5	56.1	140.3
	Capsicum campylopodium	6	98.7	39.0	41.0	96.8	73.9	74.0	100	33.3	30.0	98.0	55.6	145.0
	Capsicum hunzikerianum	8	98.2	36.3	41.0	91.0	76.0	86.0	95.9	32.0	30.1	93.8	57.2	157.1
Flexuosum	Capsicum flexuosum	5	79.7	41.3	66.4	78.4	70.5	77.4	96.7	34.0	30.0	82.0	53.0	173.8
Bolivian	Capsicum coccineum	6	83.5	41.3	61.0	95.8	87.5	8.0	94.9	32.2	30.0	87.9	42.3	99.0
Pubescens	Capsicum pubescens	7*	93.0	45.7	67.9	76.3	75.5	70.0	91.1	39.0	30.0	85.4	56.9	167.9
Purple Corolla	Capsicum eximium	4	98.5	40.4	68.0	76.3	75.2	58.5	88.4	34.4	30.5	87.8	52.2	157.0
Tovarii	Capsicum tovarii	7	90.9	37.4	199.9	82.6	68.6	73.3	76.8	30.0	29.0	87.5	44.3	302.1
Baccatum	Capsicum chacoense	4	93.8	32.7	87.3	89.7	72.8	71.8	91.7	41.7	30.0	91.9	49.3	189.0
	Capsicum praetermissum	6	97.8	32.8	75.2	91.6	77.3	61.0	97.8	40.0	30.0	95.5	50.5	166.2
	Capsicum baccatum var. baccatum	3	89.3	32.3	85.7	85.8	68.5	67.7	89.1	42.9	30.3	87.9	47.4	183.7
	Capsicum baccatum var. umbilicatum	5*	94.1	32.9	84.6	86.8	68.4	65.8	92.0	39.3	30.0	90.9	46.9	180.4
	Capsicum baccatum var. pendulum	4	91.9	33.8	82.0	92.1	66.9	70.3	91.7	39.5	29.8	91.4	47.5	182.0
Annuum	Capsicum frutescens	6*	85.4	27.7	70.5	85.3	65.3	73.0	85.3	35.0	29.5	85.3	44.8	173.0
	Capsicum chinense	6*	91.2	30.3	72.5	94.5	68.2	72.8	90.7	35.0	30.0	92.5	46.9	175.3
	Capsicum annuum var. glabriusculum	5	87.0	30.8	72.0	89.1	69.9	72.4	90.0	38.9	29.8	88.2	48.5	174.2
	Capsicum annuum var. annuum	7*	94.8	31.9	71.3	95.8	69.6	72.0	94.9	39.5	30.0	95.2	48.9	173.3

[1] 7Author for correspondence: maurograbiele@conicet.gov.ar

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Molecular characterization of the 5S rDNA non-transcribed spacer and reconstruction of phylogenetic relationships in Capsicum

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