Cytogenetics Analysis in Somatic Cells of Dermatophyllum Secundiﬂorum (Mescal Bean) Reveals Satellite Association on Subtelocentric Chromosomes

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Cytogenetics Analysis in Somatic Cells of Dermatophyllum secundiﬂorum (Mescal Bean) Reveals Satellite Association on Subtelocentric Chromosomes

Fernando Tapia-Pastrana1* and Fernando Tapia-Aguirre2

1 Division of Postgraduate Studies and Research, Faculty of Higher Studies Zaragoza, UNAM, Mexico 2 Faculty of Higher Studies Iztacala, UNAM, Mexico

Received November 16, 2020; accepted December 24, 2020

Summary A cytogenetic analysis of chromosomes in mitotic metaphase of Dermatophyllum secundiflorum, a recently synonymized species, collected in the municipality of Cardonal (Hidalgo State, Mexico) was performed. The objective was not only to obtain the karyotype, but also cytogenetic markers such as secondary constrictions and satellites. A conventional method of air drying and Giemsa staining was used in cells from root meristems which confirmed a 2n=18. The formula for the haploid karyotype was 6m+2sm+1stsat. The secondary constrictions and microsatellites were clearly located on the short arm of the st chromosomes, which differs from a previous study that placed them on metacentric chromosomes. Occasionally, metaphase plates exhibited satellite associations involving the secondary constrictions and satellites of the st chromosomes, the role of which is discussed in light of the limited information available.

Keywords Satellite association, Karyotype, Chromosome, Nucleolar organizer region, Dermatophyllum secun- diﬂorum.

Dermatophyllum Scheele is a recently synonymized population, with a special interest in the structure and genus that belongs to the Papilionoideae subfamily of behavior of SAT chromosomes as a contribution to the Leguminosae and includes six accepted species (Azani knowledge of the chromosomal architecture of a species et al. 2017). It is a genus close to the more basal clades with a wide geographical and ecological distribution be- of the papilionoid legumes known as “basal papil- longing to a recently synonymized genus. ionoids” (Pennington et al. 2001) or “early branching papilionoids” (Cardoso et al. 2012). Native to North Materials and methods America, it is distributed from the southwestern United States to central Mexico (Gandhi et al. 2011). During December 2014, fruits of D. secundiflorum D. secundiflorum (Ortega) Gandhi & Reveal is a were collected from three individuals distant from small, evergreen tree well adapted to xeric conditions each other by at least 30 m in the town of Cerritos, and widely distributed in north and south-central Mex- Municipality of Cardonal, State of Hidalgo, Mexico at ico (Still and Davies 1993, Turner 2012). The seeds rich 20°36′32.67″N and 99°07′0.3″W, and 2,010 m asl. in quinolizidine alkaloids are used by indigenous people A batch of 15 seeds was selected, five from each in the southwestern United States as a potent hallucino- individual, which were subjected to mechanical scarifi- gen in ritual practices (Izaddoost 1975). cation with garden scissors and placed to germinate in The cytogenetic studies carried out in D. secundi- Petri dishes with moistened cotton lined with absorbent florum are scarce and under the Sophora secundiflora paper in an incubator at 30°C overnight and alternat- binomial, four chromosome counts are registered that ing with 12 h at natural light. After ten days, when the agree with n=9 and 2n=18 (Bir and Kumari 1975, 1979, roots reached 2 cm in length, the meristems were sepa- Kumari and Bir 1990, Palomino et al. 1993). However, rated and pretreated with 2 mM 8-hydroxyquinoline for only the latter proposed a karyotypic formula 6m+3m- 5 h, fixed in the fixative (ethanol : acetic acid, 3 : 1), and sm, where a pair of metacentric chromosomes (m) exhib- kept there for at least 24 h. They were then treated with ited secondary constrictions and satellites on short arms. a mixture of 20% pectinase (Sigma) and 2% cellulase The present research aims to reevaluate the cytoge- (Sigma) in 75 mM KCl for 2 h at 37°C. After centrifuga- netic characteristics of D. secundiflorum in a Mexican tion at 1,500 rpm for 10 min, the cell pellet was trans- ferred to 75 mM KCl solution for 15 min at 37°C. After * Corresponding author, e-mail: [email protected] two successive rinses with the KCl solution, they were DOI: 10.1508/cytologia.86.79 again fixed in fixative and subsequently rinsed twice 80 F. Tapia-Pastrana and F. Tapia-Aguirre Cytologia 86(1) more. One or two drops of the suspension of pellet were mosome and the shortest chromosome (Range) and the placed on clean slides, air-dried, and stained in 10% Gi- longest/shortest chromosome ratio (L/S). The chromo- emsa (Hycel) for 10 min (Tapia-Pastrana and Mercado- somes were classified according to Levan et al. (1964) Ruaro 2001). Preparations were made permanent using and the index of asymmetry (TF) was obtained follow- a synthetic resin. At least ten metaphase plates of intact ing Huziwara (1962). cells with well-spread chromosomes, no chromosome overlapping, and same contraction were photographed, Results using a microscope Carl Zeiss Axioscope and analyzed for chromosome number determinations. Five photo- In total, 50 cells at the somatic metaphase of D. secun- graphs of metaphases with chromosomes having similar diflorum were observed and all exhibited 2n=18 chro- comparable degrees of contraction and centromeres mosomes (Fig. 1A–D). Figure 1E shows the karyotype clearly located were utilized to obtain the total haploid where the chromosomes were paired by homology and chromosome length (THC), mean chromosome length aligned by the centromere in decreasing order. (MC), the difference in length between the longest chro- The chromosomal complement of D. secundiflorum

Fig. 1. Chromosomes of D. secundiﬂorum. Metaphase chromosome plates showing a 2n=18. A–B: Chromosomes st with one or two satellites on short arms respectively. C–D: SA involving st chromosomes. E: Karyotype. The chromosomes are aligned in decreasing length order. *: Chromosome with secondary constriction. Scale bar=10 µm.

Table 1. Karyotypic analysis of D. secundiﬂorum.

Secondary 2n Karyotype formula THC±S.D (µm) MC±S.D (µm) Range±S.D (µm) L/S±S.D TF±S.D constriction

18 6m+2sm+1stsat 1st 17.85±2.03 1.97±0.22 0.57±0.19 1.34±0.11 38.41±0.80

THC=Total haploid chromosome length; MC=Mean chromosome length; Range=LLA-SLA; L/S=LLA/SLA. 2021 Satellite Association in Dermatophyllum secundiflorum 81 includes 12 metacentric (m) chromosomes, four sub- constriction and satellite results from the fusion of such metacentric (sm) chromosomes, and two subtelocentric structures present in sister chromatids (Fig. 1B). (st) chromosomes that carry a secondary constric- Likewise, the relationships between the numbers tion and satellite on short arms. The proposed haploid of the nucleolar organizer in the form of secondary karyotypic formula for the population under study was constrictions in plants have been widely discussed (Na- 6m+2sm+1stsat (Table 1). vashin 1928, Stack 1974, Golczyk and Joachimiak 2003, Occasionally the metaphase plates exhibited satellite Preuss and Pikaard 2007). In the Leguminosae family, a association (SA) clearly involving the lax secondary close correspondence between secondary constrictions constrictions or satellites of the st chromosome pair (Fig. and NORs has been shown: Aeschynomene spp. (Tapia- 1C–D). This phenomenon was observed in four (8%) of Pastrana et al. 2020), Crotalaria incana (Tapia-Pastrana the nuclei analyzed. et al. 2018), Erythrina americana (Tapia-Pastrana and Jiménez-Salazar 2011), Prosopis laevigata (Tapia-Pas- Discussion trana and Mercado-Ruaro 2001), Prosopis juliflora (Tapia-Pastrana 2020), Sesbania virgata (Tapia-Pastrana The diploid chromosome number for this species and Tapia-Aguirre 2018) and Tamarindus indica (Tapia- 2n=18 agrees with previous records already mentioned. Pastrana et al. 2012). Thus, we propose that in D. secun- Concerning chromosomal morphology, our results show diflorum the secondary constrictions recorded here carry that m and sm chromosomes predominate in the chromo- active NOR and this should be corroborated with the somal complements of D. secundiflorum and the asym- AgNOR staining technique (Goodpasture and Bloom metry recorded (TF%=38.39) was mainly attributed to 1975) in future studies. the presence of a st chromosome pair (r=4.46; Table 2). Regarding the associations between secondary con- In general, the chromosomes were small, and their size strictions eventually observed in metaphase nuclei, we gradually decreased with a range of 2.24 to 1.67 µm (Ta- can say that it is an event better known as SA and has ble 2). The smallest chromosome pair exhibited a clear been relatively well studied previously in humans and secondary constriction associated with satellite (Fig. other animals (Ferguson-Smith and Handmaker 1961, 1E). This type of karyotypic structure, where chromo- Evans et al. 1974, Schmid et al. 1974, Goodpasture and somes with a medium or slightly displaced centromere Bloom 1975, de Capoa et al. 1988). This phenomenon predominate and chromosomes with an displaced cen- that involves a specific position of the SAT chromo- tromere are scarce, has generally been associated with somes with their satellites directed to each other at a dis- genera of the Papilionoideae subfamily (Bairiganjan tance not greater than the width of a chromatid or con- and Patnaik 1989, Mercado-Ruaro and Delgado-Salinas nected by a region impregnated with silver was observed 1998, Souza and Benko-Iseppon 2004) and the chromo- for the first time in human mitotic chromosomes (Fer- somal complement of D. secundiflorum is no exception. guson-Smith and Handmaker 1961, Ohno et al. 1961) However, in Leguminosae, the description of karyo- and was also found on meiotic chromosomes (Ferguson- types where the sm or st chromosomes carry secondary Smith 1964). The SAT chromosomes involved carry ac- constrictions associated with satellites on their short tive NOR connected by secondary constrictions of vary- arms is increasingly frequent (Tapia-Pastrana and Mer- ing lengths that may be correlated with the frequency of cado-Ruaro 2001, Gómez-Acevedo and Tapia-Pastrana association (Henderson et al. 1973, Evans et al. 1974, 2003, Tapia-Pastrana and Gómez-Acevedo 2005, Biondo Orye 1974, Schmid et al. 1974, Miller et al. 1977). et al. 2006, Tapia-Pastrana et al. 2020). In particular, The formation of SA is attributed to the participation the detailed description of the SAT chromosomes in D. of SAT chromosomes in nucleolus formation and it has secundiflorum should consider that some metaphase been suggested that sticky nucleolar material would hold plates revealed that what appears as a single secondary the associated chromosomes together through mitosis

Table 2. Mean chromosome measures in D. secundiﬂorum.

CP TCL (µm) LLA (µm) LSA (µm) r S

1 2.24±0.25 1.30±0.11 0.93±0.15 1.41±0.16 m 2 2.14±0.29 1.24±0.18 0.89±0.13 1.39±0.14 m 3 2.05±0.25 1.18±0.12 0.86±0.14 1.38±0.15 m 4 2.05±0.27 1.37±0.23 0.68±0.07 2.01±0.30 sm 5 1.99±0.24 1.13±0.15 0.86±0.13 1.31±0.13 m 6 1.95±0.24 1.11±0.14 0.84±0.10 1.32±0.07 m 7 1.89±0.24 1.24±0.18 0.64±0.07 1.90±0.19 sm 8 1.85±0.21 1.09±0.12 0.77±0.13 1.44±0.18 m 9 1.67±0.12 1.35±0.08 0.31±0.06 4.46±0.87 st*

CP=Chromosome pair; TCL=total chromosome length; LLA=length long arm; LSA=length short arm; ±=S.D; r=arms ratio; S=shape after Levan et al. (1964). 82 F. Tapia-Pastrana and F. Tapia-Aguirre Cytologia 86(1)

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