A reference chromosomal map of the hot chili pepper <i>Capsicum pubescens</i> cv. “locoto” (Solanaceae)

Aguilera, Patricia Mabel; Debat, Humberto Julio; Seijo, José Guillermo; Grabiele, Mauro

doi:10.1590/2175-7860202172062

Patricia Mabel Aguilera ⁷7Author for correspondence: patriciamaguilera@gmail.com

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

6 These authors contributed equally to this work.

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

Humberto Julio Debat

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

6 These authors contributed equally to this work.

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

José Guillermo Seijo

Universidad Nacional del Nordeste, Instituto de Botánica del Nordeste (UNNE-CONICET) and Facultad de Ciencias Exactas y Naturales y Agrimensura, Corrientes, Argentina.

http://orcid.org/0000-0001-8699-2044

Mauro Grabiele

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

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

7Author for correspondence: patriciamaguilera@gmail.com

Area Editor: Dr. Leandro Giacomin

6
These authors contributed equally to this work.

Figures (4)
Tables (2)

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Figure 1
a-n. General morphological features of the plant and chromosome markers of Capsicum pubescens – a. detail of the plant and purple flower; b-e. fruits of cv. locoto rojo and detail of its black seeds; f. Ag-NOR stained metaphase displaying the typical 2n = 24 (arrowheads point out the satellited active-NOR pairs #10 and 12); g. DAPI stained nucleus showing heteropicnotic chromatin; h. C-DAPI fluorescent banding metaphase; note DAPI⁺ fluorescence in all the centromeres, in six interstitial and 40 distal bands -24 major-; i. CMA/DA fluorescent banding metaphase (note CMA/DA⁺ fluorescence at all the centromeres, in two interstitial and 38 distal bands -24 major-); j. same metaphase stained with DAPI/AMD (note decreased fluorescence at GC rich regions and DAPI/AMD⁺ bands at q1, q3 and q10, respectively); k. metaphase with FISH of telomeric probe (note 48 green signals at expected sites); l. metaphase with double FISH displaying 20 distal red signals of 25S and two green interstitial signals of 5S rDNA probes (asteriks) [note FISH DAPI⁺ bands at q1, q3 and q10 (arrowheads)]; m. DAPI/AMD counterstained metaphase subsequent to FISH; n. same metaphase with FISH of 25S rDNA probe. In (m.) note DAPI/AMD^- fluorescence at all the centromeres, one interstitial and 38 distal bands -24 major-, 20 of which co-localize with red rDNA signals in (n.); also note (in m.) DAPI/AMD⁺ bands at q1, q3 and q10 (arrowheads). Asterisks in h, i and m point out the 5S rDNA locus at p3, C-DAPI⁺, CMA/DA⁺ and DAPI/AMD^-, respectively. Scale bars = 10 µm; f, i-n share the scale.

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Figure 2
a-l. Characterization of heterochromatin with homology to 18S-25S rDNA in Capsicum pubescens – a-b. DAPI/AMD counterstained metaphase subsequent to FISH of 18S rDNA probe; in (a) note DAPI/AMD^- fluorescence at all the centromeres and to 38 distal bands -24 major-, 20 of which co-localize with green rDNA signals (in b); in addition, note in (a) DAPI/AMD⁺ bands at q1 and q10, pointed out with arrowheads; active-NOR pairs # 10 and 12 are hereafter indicated with their respectives numbers; c. metaphase with 20 distal green signals of FISH with IGS3 (CpIGS-A3) rDNA probe counterstained with DAPI/AMD; note DAPI/AMD⁺ bands at q1, q3 and q10 (arrowheads); d-f. double FISH of 5S rDNA probe (green) with 25S (d), IGS4 (CpIGS-B4rd) (e) and 5.8S/ITS (CpITS1,2/5.8S-1); f. rDNA probes (all the laters in red) onto metaphase (d, f) or prometaphase (e) chromosomes counterstained with DAPI; note consistently 20 distal red signals with the three probes analysed, two green interstitial signals (5S rDNA) and several FISH DAPI⁺ bands; g-i. same metaphases as in d-f, respectively, deprived of rDNA signals and colour-inverted to highligth all the FISH DAPI⁺ bands that mimic the C-DAPI banding pattern; note that each 18S-25S rDNA signal co-localized with a FISH DAPI⁺ band; asterisks point out the 5S rDNA locus at p3, FISH DAPI⁺; j-k. prometaphase with double FISH of 18S rDNA probe (green) and rDNA IGS-related probe pCp-200/33 (C33 in red), respectively; note the co-localization of 16 green and red signals, and the absence of pCp-200/33 in the active-NOR pairs #10 and 12; l. metaphase with double FISH of 18S rDNA (green) and pCp-200/33 (red) probes; note the 16 yellow signals that correspond to superimposed green and red signals of both probes and solely green signals (18S) at the active-NOR pairs #10 and 12. Arrowheads in j-l denote the 18S-25S rDNA loci at the secondary constrictions of pairs #10 and 12. Scale bars = 10 µm; a, b and l share the scale; d, f, g and i share the scale.

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Figure 3
a-h. Idiograms for each distinct cytological approach performed in Capsicum pubescens – a. C-DAPI fluorescence banding (DAPI⁺: light blue blocks); b. CMA/DA fluorescence banding (CMA/DA⁺: bright yellow blocks); c. DAPI/AMD fluorescence banding and DAPI/AMD counterstaining subsequent to FISH (DAPI/AMD^-: dark grey blocks; DAPI/AMD⁺: light blue blocks); d. DAPI counterstaining subsequent to FISH (DAPI⁺: dark blocks); e. FISH of telomeric probe (green dots); f. Ag-NOR staining (active-NOR: dark brown blocks); g. double FISH of 5S rDNA probe (green dots) and all the probes of the 18S-25S rDNA (red-green barred blocks) assayed except pCp-200/33; h. FISH of pCp-200/33 probe (red blocks). Polymorphic 18S-25S and pCp-200/33 rDNA loci at p2, q3 and q11 are coloured in a paler tone. Scale bar = 5 µm.

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Figure 4
Combined chromosomal map of repetitive loci and classical cytological landmarks in Capsicum pubescens. Short chromosome arms are clockwise oriented. HCL = haploid complement length; RL% = relative length of chromosomes expressed as percentage; C = centromeric index. Size of each chromosome length (centromere-telomere) is expressed in µm. Estimated whole chromosome length is expressed in Mbp.

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Table 1
Cytogenetic markers identified in Capsicum pubescens cv. “locoto”. Het = heterochromatinized; p = short chromosome arm; q = long chromosome arm; D = distal; I = interstitial; C = centromeric. Numbers between brackets refer to chromosome pairs. + = increased fluorescence; - = decreased fluorescence; a = according to idiogram of Fig. 4; b = sometimes fused to distal band at q3; c = all the probes of the 18S-25S rDNA assayed except pCp-200/33; d = polymorphic loci.

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Table 2
Heterochromatin amount, type and distribution in somatic chromosomes of Capsicum pubescens cv. “locoto”. Chr = chromosome; Het = heterochromatin; a = values are expressed as percentage of the chromosome size; b = includes all types of Het detected with different fluorochromes.

Technique	Revealed marker	Number and position of loci^a
C-DAPI fluorescent banding	Total heterochromatin⁺	20 D (p1-4, 6-12; q2-6, 8-11); 3 I (q1; p3; q3^b); 12 C
CMA/DA fluorescent banding	GC-rich heterochromatin⁺	19 D (p1-4, 6-12; q2-6, 8, 9, 11); 1 I (p3); 12 C
DAPI/AMD fluorescent banding	GC-rich heterochromatin‒ AT-rich heterochromatin⁺	19 D (p1-4, 6-12; q2-6, 8, 9, 11); 1 I (p3); 12 C 1 D (q10); 2 I (q1, 3)
DAPI/AMD subsequent to FISH	GC-rich heterochromatin‒ AT-rich heterochromatin⁺	19 D (p1-4, 6-12; q2-6, 8, 9, 11); 1 I (p3); 12 C 1 D (q10); 2 I (q1, 3)
DAPI subsequent to FISH	Total heterochromatin⁺	20 D (p1-4, 6-12; q2-6, 8-11); 3 I (q1; p3; q3^b); 12 C
FISH of telomeric probe	Telomeric sequence	24 D (overall chromosome complement)
Ag-NOR staining	Active-NOR	2 D (p10, 12)
FISH of 18S-25S rDNA probes^c	Total 18S-25S rDNA	13 D (p1, 2^d, 3, 6, 10, 12; q2, 3^d, 4-6, 8, 11^d)
FISH of pCp-200/33 probe	Het 18S-25S rDNA	11 D (p1, 2^d, 3, 6; q2, 3^d, 4-6, 8, 11^d)
FISH of 5S rDNA probe	5S rDNA	1 I (p3)

Chr	Size µm	Size Mbp	GC rich active-NOR 18S-25S rDNA^a	GC rich Het of inactive 18S-25S rDNA^a	GC rich Het unrelated to rDNA^a	5S rDNA^a	AT rich Het^a	Total Het^ab	Euchromatin/Het^b ratio
1	7.75	419.28	-	17.68	-	-	3.87	24.77	3.04
2	7.50	405.75	-	23.60	-	-	-	26.93	2.71
3	7.50	405.75	-	27.20	-	3.33	4.00	37.87	1.64
4	7.05	381.41	-	19.72	3.55	-	-	26.81	2.73
5	7.00	378.70	-	13.71	-	-	-	17.29	4.79
6	6.90	373.29	-	36.81	-	-	-	40.43	1.47
7	6.80	367.88	-	-	3.68	-	-	7.35	12.60
8	6.55	354.36	-	18.93	3.82	-	-	26.56	2.76
9	6.50	351.65	-	-	7.69	-	-	11.54	7.67
10	6.40	346.24	12.97	-	-	-	5.47	22.34	3.48
11	6.25	338.13	-	16.00	4.00	-	-	24.00	3.17
12	5.50	297.55	11.09	-	-	-	-	15.64	5.40

[1] 7Author for correspondence: patriciamaguilera@gmail.com

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Brasil

A reference chromosomal map of the hot chili pepper Capsicum pubescens cv. “locoto” (Solanaceae)

Abstract