elocation-id: e3733
The research work was developed at the Plant Genetic Resources Laboratory of the ‘Presidente Juárez’ Faculty of Agrobiology of the Michoacán University of San Nicolás de Hidalgo, in 2019 and 2022. The aim of this research was to evaluate the usefulness of functional markers derived from CYP450 for genetic diversity studies in Capsicum pubescens. The genetic material consisted of 31 cultivated varieties of C. pubescens from three different localities in the state of Michoacán, Mexico. Genomic DNA was obtained based on the protocol of Huang et al. (2013) and two combinations of CYP450 primers were included in the analysis. The amplification products were separated on 8% acrylamide gels and stained with silver nitrate. A total of 85 loci were detected: the CYP2B6F/CYP2C19R combination detected 34 polymorphic loci, while the CYP2C19F/CYP1A1R combination detected only 27. The diversity analysis of C. pubescens identified 1.54 alleles per locus, 1.33 effective number of alleles per locus, Shannon index of 0.3, a heterozygosity index of 0.2 and 60.39% of polymorphic loci. The results obtained show that markers derived from CYP450 are an efficient and low-cost alternative for studies of genetic diversity in plant species.
Capsicum pubescens, genetic variability.
The species C. pubescens, commonly known as perón, manzano, canario, cera or rocoto pepper (Escalera-Ordaz, 2019), is native to the highlands of South America, flourishing in elevated areas between 1 200 and 3 000 masl. In Mexico, it is found in the states of Mexico, Puebla, Veracruz, Michoacán, and Chiapas (Aguirre and Muñoz, 2015).
This species has a diversity of uses: medicinal, it is consumed fresh, dehydrated, or as canned food and is a source of natural dyes (Escalera-Ordaz, 2019).
For greater understanding, studies have been carried out on genetic diversity, population structure and phylogenetic relationships. In this sense, molecular markers are considered an important biotechnological tool for the evaluation of genetic diversity thanks to the characteristics they possess (Gil-Langarica, 2008). Among the techniques that have been used are AFLPs (Vos et al., 1995) and microsatellite sequences such as ISSRs (Zietkiewiez et al., 1994) and SRAPs (Li and Quiros, 2001).
However, these techniques currently have certain disadvantages that can become an obstacle: the requirement of large amounts of DNA (Rentería, 2007), reproducibility (Ríos et al., 2009) and prior knowledge of DNA sequences, among others (Carvalho et al., 2015). Research on this species has been mainly limited to varieties, hybrids, and lines of different Capsicum species (Castañón-Nájera et al., 2011; Contreras-Toledo et al., 2011; Mahmoud, 2013; Carvalho et al., 2015; Toledo-Aguilar et al., 2016; Xiao-min et al., 2016; López-Espinosa et al., 2018). In contrast, C. pubescens has a lower level of research (Pardey and García, 2011; Lijun and Xuexiao, 2012).
Cytochrome CYP450 was identified in 1958 as a cellular pigment (Jaimes-Santoyo et al., 2014), it is found in low concentrations in different plant organs and in some cellular organelles such as endoplasmic reticulum, plasma membrane, vacuole, mitochondria, and Golgi apparatus. In this regard, different cytochrome CYP450 molecules have been identified in a single organism (Valencia-Quintana et al., 2009) with different functions within plants: synthesis of various compounds of defense against insects and pathogens, in the biosynthesis of gibberellins, as well as in the process of fruit ripening.
In Arabidopsis, 270 genes belonging to 45 different families of CYP450 have been identified (Gonzáles-Mendoza, 2009), making CYP450 one of the largest families of enzymatic proteins in plants (Bak, 2011). Genes of the CYP88A family have also been isolated from Cucurbita maxima (Helliwell et al., 2000) and Zea mays (Winkler and Helentjaris, 1995), of the CYP73A9v1 family from Pisum sativum (Whitbred and Schuler, 2000) and CYP71A1 from Persea americana.
Functional genomic markers based on CYP450 (Shakeel et al., 2019) were originated from a study that evaluated the polymorphism detected by CYP450 in mammals and subsequently used as universal tools to assess the genetic diversity of plant species. Around 51 species have been evaluated with this marker (Yamanaka et al., 2003) and successfully in different plant species: Musa spp. (Wan et al., 2005), Withania coagulans (Gilani et al., 2009), Curcuma amada (Shakeel et al., 2019), Eleusine coracana (Panwar et al., 2010), Moringa oleifera (Saini et al., 2013), Oryza sativa (Yamanaka et al., 2011), Sechium edule (Machida-Hirano et al., 2015), among others.
The polymorphism levels determined range from 0.28% to 88.25%. Given the characteristics and results obtained with the CYP450 marker in studies of genetic diversity in different species, it was proposed to use them for preliminary studies of genetic variability in cultivated varieties of C. pubescens.
In the present research, 31 cultivated varieties of C. pubescens were used: 11 of them from the municipality of Tingambato (TIN) and 20 from two localities in the municipality of Uruapan: 11 from Toreo el Bajo (TOB) and 9 from Tiamba (TIA). The sites of origin of the analyzed materials range between 1 623 and 2 282 masl.
DNA was obtained based on the procedure described by Huang et al. (2013). Some modifications made consisted of the lyophilization of leaf tissue without the previous use of liquid nitrogen and the substitution of chloroform and isoamyl alcohol with dichloromethane (CH2Cl2) and ethanol, respectively. The concentration of the isolated DNA was determined with the help of a NanoDrop 2000c (Thermo Scientific®).
To obtain amplicons, 10 μl of the following PCR reaction mixture were used: 2.5 μl ddH2O, 0.3 mM MgCl, 0.6 μM of each primer, 3.5 μl 2X Taq network (0.7 X) and 2 μl of a DNA solution (25 ng μl-1). The oligonucleotides used were two combinations of cytochrome CYP450-derived primers: CYP2B6F (5’ gac tct tgc tac tcc tgg gtt 3’)/CYP2C19R (3’ cca tcg att ctt ggt gtt ct 5’) and CYP2C19F (5’ tcc ttg tgc tct gtc tct ca 3’)/CYP1A1R (3’ aag gac atg ctc tga cca tt 5’) (Inui et al., 2000). For amplification, a 3 PrimeG techne® thermal cycler was used.
For the combination of CYP2B6F/CYP2C19R oligos, it was as follows: an initial stage of 5 min at 94 °C, followed by 35 amplification cycles as follows: denaturation 60 s at 94 °C, hybridization 60 s at 47 °C and extension 60 s at 72 °C. For the combination of CYP2C19F/CYP1A1R oligos, hybridization was performed for 60 s at 54 °C. For both combinations, a final extension of 10 min at 72 °C was included. The amplicons were separated by 8% acrylamide gel electrophoresis in 200 ml vertical systems (EnduroTM Power Supplies 300V).
The molecular reference markers were 20 bp and 100 bp. The detection of the amplified products was carried out by silver nitrate staining and they were visualized on a Carestream® Gel Logic 112 imaging system (Sanguinetti and Simpson, 1994).
For this procedure, three groups of varieties were organized, considering the place of origin as a grouping criterion. Data were analyzed with the GenAlex statistical package (Peakall and Smouse, 2012) to calculate genetic diversity parameters: number of alleles (N), average number of alleles/locus (Na), effective number of alleles (Ne), Shannon index (I), heterozygosity (He) and percentage of polymorphic loci (% P). From a matrix of averages, the matrix of genetic distances between varieties was calculated and the clustering analysis was performed with the Neighbor-Joining method. The dendrogram was generated using the MEGA5 program (Tamura et al., 2011).
Determined level of polymorphism. The combinations of markers used generated bands between 40 and 500 bp. This contrasts with what other authors (Mahmoud, 2013) have obtained in studies carried out on C. annuum; in the latter case, they used codominant markers of the ISSR type. A total of 85 loci were detected, the CYP2B6F/CYP2C19R combination revealed 34 polymorphic bands, while the CYP2C19F/CYP1A1R combination revealed 27 polymorphic bands, for a polymorphism level of 60.39%, this value was 8.83% higher on average than that determined in other Capsicum species and 29.14% higher than that reported (Pardey and García, 2011) for C. pubescens using SSRs.
In general, CYP450 combinations detected a higher level of polymorphism, 41.9% and 35.59% higher than that detected with ISSRs (Lijun and Xuexiao, 2012) and RAPDs (Bobadilla-Larios et al., 2017), respectively. In contrast, studies on varieties of C. frutescens and C. annuum (Castañón-Nájera et al., 2011) and on landrace populations of C. chinense (López-Espinosa et al., 2018) have reported average polymorphism levels of 95.4% using ISSR and AFLP markers.
It is worth highlighting the level of polymorphism (60.39%) determined in C. pubescens compared to the average (20.91%) detected with other markers (Pardey and García, 2011; Lijun and Xuexiao, 2012) in the same species. On the other hand, there are few studies on the use of markers derived from CYP450 in other plant species, with those carried out in Musa ssp. and in Curcuma amada (Shakeel et al., 2019) standing out, where polymorphisms of 65.2% and 94.6%, respectively, have been determined.
This type of markers is based on a multigene family that records diversity in functional regions of the genome, which explains the levels of polymorphism obtained and, therefore, it has been used to characterize the genetic diversity and variability of species of insects (Giraldo et al., 2011), hoofed mammals, and cetaceans (Irwin et al., 1991).
In addition, genes derived from CYP450, which are widely distributed within the plant genome, can be used universally; however, their use in plant species (Yamanaka et al., 2003; Wan et al., 2005; Gilani et al., 2009; Panwar et al., 2010; Yamanaka et al., 2011; Saini et al., 2013; Machida-Hirano et al., 2015; Shakeel et al., 2019) has been limited.
Genetic diversity analysis. The analysis identified an average of 1.54 alleles per locus, 1.33 effective number of alleles per locus, a Shannon index of 0.3, a heterozygosity index of 0.2 and a percentage of polymorphic loci of 60.39% (Table 1). The estimated heterozygosity value was low (0.2) compared to those reported by Xiao-min et al. (2016); Toledo-Aguilar et al. (2016), where values ranging from 0.36 to 0.59 were determined. The heterozygosity determined in C. annuum (Contreras-Toledo et al., 2011), C. frutescens and C. chinense (Carvalho et al., 2015; López-Espinosa et al., 2018) with SSR markers yielded average values of 0.42, higher than those reported in this research.
| Populations | Na | Nab | Nec | Id | Hee | Pf (%) |
|---|---|---|---|---|---|---|
| Toreo el Bajo | 11 | 1.66 | 1.38 | 0.34 | 0.23 | 67.06 |
| Tingambato | 11 | 1.55 | 1.31 | 0.29 | 0.19 | 60 |
| Uruapan | 9 | 1.42 | 1.3 | 0.27 | 0.18 | 54.12 |
| Mean | 10.33 | 1.54 | 1.33 | 0.3 | 0.2 | 60.39 |
Similar results have been obtained with the use of AFLPs (Guzmán et al., 2005) and ISSRs (Lijun and Xuexiao, 2012). It has also been observed that when the indicated species are related to C. pubescens, low values of genetic diversity are obtained (Pardey and García, 2011).
CYP450 functional markers have shown their feasibility for estimating high levels of genetic diversity, even in some plant species such as Musa spp. (Wan et al., 2005) and Oryza sativa (Yamanaka et al., 2011). In this sense, the He values determined in the populations studied in the three localities were similar, with an average of He= 0.2. This agrees with the levels of polymorphism estimated in each of the populations indicated.
The estimation of the Shannon diversity index showed that the richness of individuals in the population of Uruapan was lower (I= 0.27) compared to the populations of Tingambato and Toreo el Bajo (I= 0.29 and I= 0.34, respectively). The estimated average value for this parameter was I= 0.3 and may be a function of the homogeneity of the population and the frequency of alleles (Glasenapp et al., 2015). The analyses of genetic diversity parameters obtained with CYP450 are similar (Machida-Hirano et al., 2015) to those generated with other types of markers such as SSRs and RAPDs.
Kinship relationships. In Figure 1, it was observed that the markers derived from CYP450 identified two perfectly defined groups, both groups were not related to the morphological characteristics of the fruits, but neither to their place of origin. A polytomy was observed in the dendrogram since there is not enough information to explain this relationship (Martínez, 2007). The formation of groups based on the polymorphism detected with CYP450-derived markers has not been explained (Wan et al., 2005; Gilani et al., 2009; Saini et al., 2013), it is likely that increasing the number of primers will lead to more information about these types of clusters (Wan et al., 2005).
In both groups, those that stand out for their greater genetic similarity and based on the sequences analyzed are the varieties from the locality of Tiamba: in group 1 the TIA4 with TIA5, and in group 2 the variety TIA1 with TIA2. However, in group 1 there is a predominance of cultivated varieties from Tingambato, while in group 2 those from the locality of Toreo predominate. The formation of groups based on the polymorphism detected with CYP450-derived markers has not been explained (Wan et al., 2005; Gilani et al., 2009; Saini et al., 2013), it is very likely that increasing the number of primers will lead to more information about this type of clusters.
The use of these markers in mammals (Giraldo et al., 2011) and in the identification of analogous sequences of CYP450 genes in different plant species (Gonzáles-Mendoza, 2009; Bak, 2011) allowed CYP450 to be considered as a universal tool for the assessment of genome genetic diversity in diverse plant species that do not have relevant genetic markers.
These types of markers represent an ideal system (Yamanaka et al., 2011) to reveal the genetic diversity present in individuals, populations and species, since they allow the detection of a high level of polymorphism, are cost-effective, with limited budgets, without significant differences in terms of group formation, but useful in the estimation of genetic diversity and rapid assay (Panwar et al., 2010), this makes them a reliable alternative for studies of genetic diversity (Shakeel et al., 2019) in plant species.
The results generated in this research work, in cultivated varieties of C. pubescens, show the usefulness of functional markers derived from CPY450 in studies to generate information quickly and inexpensively on the variability and genetic diversity of individuals or populations of closely related plant species.
To the Coordination of Scientific Research of the Michoacán University of San Nicolás de Hidalgo (CIC-UMSNH, for its acronym in Spanish) for the partial funding of the project and to the National Council of Humanities, Sciences and Technologies (CONAHCYT, for its acronym in Spanish) for the scholarship for a postdoctoral stay.
Aguirre, H. E. y Muñoz, O. V. 2015. El chile como alimento. Revista Ciencia. 66(3):16-23. https://www.revistaciencia.amc.edu.mx/images/revista/66-3/PDF/Chile.pdf.
Bak, S.; Beisson, F.; Bishop, G.; Hamberger, B.; Höfer, R.; Paquette, S. and Werck-Reichhart, R. D. 2011. Cytochromes P450. The Arabidopsis Book. 9:2-56. https://doi.org/10.1199/tab.0028.
Bobadilla-Larios, V.; Esparza-Ibarra, E.; Delgadillo-Ruiz, L.; Gallegos-Flores, P. y Ayala-Lujan, J. L. 2017. Variedades de chile (Capsicum annuum L.) identificadas mediante marcadores RAPD. Tropical and Subtropical Agroecosystems. 20(3):465-473. https://www.redalyc.org/pdf/939/93953814014.pdf.
Carvalho, S. I. C.; Ragassi, C. F.; Oliveira, I. B.; Amaral, Z. P. S.; Reifschneider, F. J. B.; Faleiro, F. G. and Buso, G. S. C. 2015. Transferability of microsatellite markers of Capsicum annuum L. to C. frutescens L. and C. chinense Jacq. Genetics and Molecular Research. 14(3):7937-7946. https://doi.org/10.4238/2015.July.17.1.
Castañón-Nájera, G.; Ramírez-Meraz, M.; Ruiz-Salazar, R. y Mayek-Pérez, N. 2011. Aplicación de marcadores AFLP para explorar heterosis en Capsicum spp. Revista Internacional de Botánica Experimental. 80(1):53-58. http://www.revistaphyton.fundromuloraggio.org.ar/vol80/Castanon-Najera-2011.pdf.
Contreras-Toledo, A. R.; López-Sánchez, H.; Santacruz-Varela, A.; Valadez-Moctezuma, E.; Aguilar-Rincón, V. H.; Corona-Torres, T. y López, P. A. 2011. Diversidad genética en México de variedades nativas de chile “poblano” mediante microsatélites. Revista Fitotecnia Mexicana. 34(4):225-232. http://www.scielo.org.mx/scielo.php?pid=S018773802011000400003yscript=sci-arttext.
Escalera-Ordaz, A. K. y Guillén-Andrade, H. 2019. Formas y colores del chile perón. Revista Saber Más. 48(8):42-44. https://www.sabermas.umich.mx/archivo/ articulos/416-numero-48/786-chile-peronvariabilidad-de-formas-y-colores.html.
Gilani, A. S.; Kikuchi, A. and Watanabe, K. N. 2009. Genetic variation within and among fragmented populations of endangered medicinal plant, Withania coagulans (Solanaceae) from Pakistan and its implications for conservation. African Journal of Biotechnology. 8(13):2948-2958. http://www.academicjournals.org/AJB/PDF/ pdf2009/6%20Jul/Gilani%20et%20al.pdf.
Giraldo, H. P. A.; Uribe, S. S. I. y López, R. A. 2011. Análisis de secuencias de ADN mitocondrial (Cytb y ND1) en Lucilia eximia (Diptera: Calliphoridae). Revista Colombiana de Entomología. 37(2):273-278. http://www.scielo.org.co/pdf/rcen/v37n2/v37n2a20.pdf .
Glasenapp, J. S.; Frieden, B. R. and Cruz, C. D. 2015. Shannon mutual information applied to genetic systems. Quantitative Biology. 1(1):1-20. https://arxiv.org/ftp/arxiv/papers/1512/1512.02324.pdf.
Gonzáles-Mendoza, D. 2009. El complejo enzimático citocromo p450 en las plantas. Revista internacional de contaminación ambiental. 23(4):177-183. http://www.scielo.org.mx/pdf/rica/v23n4/v23n4a3.pdf.
Guzmán, F. A.; Ayala, H. D.; Azurdia, C. A.; Duque, M. C. and De Vicente, M. C. 2005. AFLP assessment of genetic diversity of Capsicum genetic resources in Guatemala: Home gardens as an option for conservation. Crop Science. 45(1):363-370. http://qualquant.org/wp-content/uploads/ethnoecology/2005%20 Guzman363.pdf.
Huang, Q. X.; Wang, X. C.; Kong, H.; Guo, Y. L. and Guo, A. P. 2013. An efficient DNA isolation method for tropical plants. African Journal of Biotechnology . 12(19):2727-2732. https://doi.org/10.5897/AJB12.524.
Jaimes-Santoyo, J.; Montesinos-Sampedro, A.; Barbosa-Cobos, R. E.; Moreno-Mutio, S. G.; Rodríguez-Ballesteros, D.; Ramos-Cervantes, T.; Ocharán-Hernández, M. E.; Toscano-Garibay, J. y Beltrán-Ramírez, O. 2014. El citocromo P-450. Revista del Hospital Juárez de México. 81(4):250-256. https://www.medigraphic.com/pdfs/juarez/ju-2014/ju144j.pdf.
Lijun, O. and Xuexiao, Z. 2012. Inter simple sequence repeat analysis of genetic diversity of five cultivated pepper species. African Journal of Biotechnology . 11(4):752-757. https://doi.org/10.5897/AJB10.2551.
López-Espinosa, S. T.; Latournerie-Moreno, L.; Castañón-Nájera, G.; Ruiz-Sánchez, E.; Gómez-Leyva, J. F.; Andueza-Noh, R. H. y Mijangos-Cortés, J. O. 2018. Diversidad genética de chile habanero (Capsicum chinense jacq.) mediante ISSR. Revista Fitotecnia Mexicana . 41(3):227-236. https://doi.org/10.35196/rfm.2018.3.227-236.
Machida-Hirano, R.; Cortés-Cruz, M.; González, B. A. A.; Íñiguez, J. C.; Shirata, K. and Watanabe, K. N. 2015. Isolation and characterization of novel microsatellite markers in chayote [Sechium edule (Jacq.) Sw.]. American Journal of Plant Sciences. 6(13):2033-2041. https://doi.org/10.4236/ajps.2015.613203.
Martínez, C. L. 2007. Reconstrucción de la historia de cambio de los caracteres. Ed. Ecología Molecular. 87-152 pp. https://www.researchgate.net/publication/ 258129643-Ecologia-Molecular.
Panwar, B.; Saini, R. K.; Sharma, N.; Yadav, D. and Kumar, A. 2010. Efficiency of RAPD, SSR and Cytochrome P450 gene-based markers in accessing genetic variability amongst finger millet (Eleusine coracana) accessions. Molecular Biology Reports. 37 https://doi.org/10.1007/s11033-010-0067-5.
Pardey, R. C. y García, D. M. A. 2011. Caracterización molecular de 135 introducciones de Capsicum procedentes del banco de germoplasma de la Universidad Nacional de Colombia sede Palmira. Revista Intropica. 6(1):21-32. https://agris.fao.org/agris-search/search.do?recordID=DJ20220180543.
Peakall, R. and Smouse, P. E. 2012. GenAlex 6.5: Genetic analysis in Excel. Population genetic software for teaching and research-an update. Bioinformatics. 28(19):2537-2539. https://doi.org/10.1093/bioinformatics/bts460.
Rentería, A. M. 2007. Breve revisión de los marcadores moleculares. Ed. Ecología Molecular. 541-571. https://hopelchen.tecnm.mx/principal/sylabus/fpdb/ recursos/r119349.PDF.
Saini, R. K.; Saad, K. R.; Ravishankar, G. A.; Giridhar, P. and Shetty, N. P. 2013. Genetic diversity of commercially grown Moringa oleifera Lam. cultivars from India by RAPD, ISSR and cytochrome P 450-based markers. Plant Systematics and Evolution. 299(7):1205-1213. https://doi.org/10.1007/s00606-013-0789-7.
Shakeel, A. J.; Kikuchi, A.; Ahmad, D. and Watanabe, K. N. 2019. Characterization of the genetic structure of mango ginger (Curcuma amada Roxb.) from Myanmar in farm and genebank collection by the neutral and functional genomic markers. Electronic Journal of Biotechnology. 13(6):1-11. ISSN: 0717-3458.
Tamura, K.; Peterson, D.; Peterson, N.; Stecher, G.; Nei, M. and Kumar, S. 2011. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Molecular Biology and Evolution. 28(10):2731-2739. https://doi.org/10.1093/molbev/msr121.
Toledo-Aguilar, R.; López-Sánchez, H.; Santacruz-Varela, A.; Valadez-Moctezuma, E.; López, P. A.; Aguilar-Rincón, V. H.; González-Hernández, V. A. and Vaquera-Huerta, H. 2016. Characterization of genetic diversity of native ‘Ancho’ chili populations of Mexico using microsatellite markers. Chilean Journal of Agricultural Research. 76(1):18-26. https://doi.org/10.4067/S0718-58392016000100003.
Vos, P.; Hogers, R.; Bleeker, M.; Reijans, M.; Lee, T.; Van De, Hornes, M. Friters, A.; Pot, J; Paleman, J.; Kuiper, M. and Zabeau, M. 1995. AFLP: A new technique for DNA fingerprinting. Nucleic Acids Research. 23(21):4407-4414. https://doi.org/10.1093/nar/23.21.4407.
Wan, Y.; Watanabe, J. A.; San, S. Y.; Htaik, T.; Win, K.; Yamanaka, S.; Nakamura, I. and Watanabe, K. N. 2005. Assessment of genetic diversity among the major Myanmar banana landraces. Breeding Science. 55(3):365-369. https://www.jstage.jst.go.jp/article/jsbbs/55/3/55-3-365/-pdf.
Xiao-min, Z.; Zheng-hai, Z.; Xiao-zhen, G.; Sheng-li, M.; Xi-xiang, L.; Alain, J. C. P.; Li-Hao, W. and Bao-xi, Z. 2016. Genetic diversity of pepper (Capsicum spp.) germplasm resources in China reflects selection for cultivar types and spatial distribution. Journal of Integrative Agriculture. 15(9):1991-2001.
Yamanaka, S.; Suzuki, E.; Tanaka, M.; Takeda, Y.; Watanabe, J. A. and Watanabe, K. N. 2003. Assessment of cytochrome P450 sequences offers a useful tool for determining genetic diversity in higher plant species. Theoretical and Applied Genetics. 108(1):1-9. https://doi.org/10.1007/s00122-003-1403-0.