Biotecnología

 

Comparative study of the discriminating capacity of DNA markers and their effectiveness in establishing genetic relationships in the genus Tigridia

 

Estudio comparativo de la capacidad de discriminación de marcadores de ADN y su efectividad en el establecimiento de relaciones genéticas en el género Tigridia

 

Jesús I. Reyes-Díaz¹, Amaury M. Arzate-Fernández^1*, José L. Piña-Escutia¹, Luis M. Vázquez-García²

 

¹ Centro de Investigación y Estudios Avanzados en Fitomejoramiento, Facultad de Ciencias Agrícolas, Universidad Autónoma del Estado de México. Carretera Toluca-Ixtlahuaca km 11.5, Campus Universitario El Cerrillo. 50200. Toluca, Estado de México, México. *Author for correspondence. (amaury1963@yahoo.com.mx).

² Centro Universitario Tenancingo, Universidad Autónoma del Estado de México, Carretera Tenancingo-Villa Guerrero Km. 1.5, Ex hacienda de Santa Ana. 52400. Tenancingo, Estado de México, México.

 

Received: November, 2014.
Approved: April, 2015.

 

Abstract

Tigridia Jussieu is an endemic genus to Mexico and taxonomically difficult with limited information about its genetic variability. A diversity assessment conducted using different DNA markers as an inter simple sequence repeat (ISSR) and random amplified polymorphic DNA (RAPD) markers will be helpful in the establishment of a broad-based description for improved germplasm curation and the identification of germplasm for genome mapping and breeding of these species. Thus, the objective of this study was to characterize 15 wild species of Tigridia by using RAPD and ISSR molecular markers. This study was carried out in the laboratory of Plant Molecular Biology at the Faculty of Agricultural Sciences of the Universidad Autónoma del Estado de México between August and November of 2011. In this assay, 13 RAPD primers of 10, 15 and 20 b, and five ISSR primers of the anchored type (ASSR) of 17 b were used to assess the level of genetic variation among 15 wild species of Tigridia. With both markers there were 163 amplified bands of which 150 (92.02 %) were polymorphic. The RAPD primers of 10 b generated 12 specific bands with a polymorphism of 95.12 %, for 15 b primers those values were five and 82.93 %, and for 20 b primers eight and 94.59 %, respectively. The RAPD pooled primers presented a polymorphism of 90.76 %, the genetic distance (Gd) among the species ranged from 0.16 (between T. illecebrosa and T. huajuapanensis) to 0.57 (between T. multiflora and T. augusta). The ISSR primers showed more polymorphism (95.45 %) than RAPD primers. With ASSR primers the highest genetic association (Gd = 0.89) was observed between T. mexicana ssp. mexicana and T. durangense, whereas the least related were T. vanhouttei spp. vanhouttei and T. multiflora (Gd=0.14). This study shows that 10 base random primers and 17 base anchored primers were more efficient to detect polymorphism and genetic differentiation among Tigridia species.

Key words: Iridaceae, RAPD, ASSR, molecular characterization.

 

Resumen

Tigridia Jussieu es un género endémico de México, su taxonomía es difícil, y la información sobre su variabilidad genética es limitada. Una evaluación de la diversidad usando diferentes marcadores de ADN, como marcadores de inter-secuencias simples repetidas (ISSR) y amplificación aleatoria de ADN polimórfico (RAPD), será útil para establecer una descripción de base amplia, para la conservación de germo-plasma mejorado y la identificación de germoplasma para el mapeo del genoma y el cultivo de estas especies. Así, el objetivo de este estudio fue caracterizar 15 especies silvestres de Tigridia usando marcadores moleculares RAPD e ISSR. Este estudio se realizó en el Laboratorio de Biología Molecular Vegetal de la Facultad de Ciencias Agrícolas de la Universidad Autónoma del Estado de México, entre agosto y noviembre de 2011. En este estudio, 13 iniciadores RAPD de 10, 15 y 20 b y cinco iniciadores ISSR de tipo anclado (ASSR) de 17 b fueron usados para evaluar el nivel de variación genética entre las 15 especies silvestres de Tigridia. Con ambos marcadores se tuvieron 163 bandas amplificadas, de las cuales 150 (92.02%) eran polimórficas. Los iniciadores RAPD de 10 b generaron 12 bandas específicas con un polimorfismo de 95.12 %, para los iniciadores de 15 b esos valores fueron cinco y 82.93 % y ocho y 94.59 % para los de 20 b, respectivamente. Los iniciadores RAPD agrupados presentaron un polimorfismo de 90.76 %, la distancia genética (G_D) entre las especies varió de 0.16 (entre T. illecebrosa y T. huajuapanensis) a 0.57 (entre T. multiflora y T. augusta). Los iniciadores ISSR mostraron más polimorfismo (95.45 %) que los iniciadores RAPD. Con los iniciadores ASSR la asociación genética mayor (G_D=0.89) se observó entre T. mexicana ssp. mexicana y T. durangense, mientras que los menos relacionados fueron T. vanhouttei spp. vanhoutteiy T. multiflora (G_D = 0.14). Este estudio mostró que los iniciadores aleatorios de 10 bases y los iniciadores anclados de 17 bases fueron más eficientes para detectar el polimorfismo y diferenciación genética entre la especies de Tigridia.

Palabras clave: Iridaceae, RAPD, ASSR, caracterización molecular.

 

INTRODUCTION

In Mexico there is a large biodiversity of species; among those, Tigridia species are culturally significant (medicinal, ornamental, ceremonial and food purposes) and they represent one of the most important biotic resources for the Mexican people (Vázquez et al., 2001). The genus Tigridia Jussieu (Tigridieae: Iridaceae) is distributed around the world and Mexico is the focal point for its diversity. The irregular topography and climatic diversity have generated a high number of endemics in this group of plants (Rodríguez and Ortiz-Catedral, 2003b). The Tigridia genus group comprises about 45 species and in Mexico there are about 36 species and six subspecies, of which 29 species and six subspecies are endemic. These plants have colourful flowers that exhibit great morphological variation, making many species potentially valuable as cultivated plants. Also, Tigridia is considered a taxonomically difficult genus since its useful floral characteristics, which defines the boundaries among species, cause difficulties for the specimen analysis (Rodríguez and Ortiz-Catedral, 2003a). Furthermore, the morphological analysis can contain inaccurate information because it is based on characteristics highly influenced by the environment (Vidal-Barahona et al., 2006).

Thus, the characterization and conservation of wild plant genetic resources should be essential practices that allow the protection and conservation of a plant genetic heritage, especially those of endemic species. Likewise, this allows to evaluating the adaptation of these species to climatic or anthropogenic changes and contribute to the knowledge of the genetic variability of each species and genus, facilitating the selection of genotypes for the development of protection programs or genetic breeding (Gutiérrez-Diez et al., 2009; Valdés et al., 2010).

Traditionally, the study of genetic variation and identification of plant species consisted of the morphological description of germplasm. Now, biochemical and molecular markers techniques offer new tools to study the genetic variability of plant populations at DNA levels, making their characterization more specific. Though there is a great number of DNA markers due to low technical input and low cost, Random Amplification of Polymorphic DNA (RAPD), or Inter Simple Sequence Repeat (ISSR), and Anchored Simple Sequence Repeat (ASSR) primers (Debenner and Mattiesch, 1998; Rentaría, 2011), are applicable to any type of plant material without the need to evaluate many characters and, besides, free from epistatic effects (Azofeifa-Delgado, 2006). The success of RAPD is highly dependent on these small arbitrary oligonucleotides that hybridize onto the complementary DNA fragments. Its performance is convenient and does not require any information about the DNA sequence to be amplified (Weder, 2002). The ISSR technique amplifies genomic regions between two microsatellites with ASSR type primers (Yamagishi et al., 2002) and it is fast because of its high rate of reproducibility and efficiency while detecting polymorphisms (Pradeep et al., 2002). Both techniques are successfully utilized to calculate the intra or inter-specific genetic diversity in different domesticated and wild species, like Lilium maculatum (Arzate-Fernández et al., 2005), nine varieties of Tigridiapavonia (Piña-Escutia et al., 2010a, 2010b) and Sprekelia formosissima (Bautista-Puga et al., 2011).

Thus, the objective of this study was to characterize 15 wild species of Tigridia using RAPD and ISSR molecular markers.

 

MATERIALS AND METHODS

Plant material

Fifteen wild species of Tigridia Jussieu were used in the present analyses: T. alpestris ssp. obtusa, T. augusta, T. bicolor, T. durangense, T. ehrenbergii ssp. ehrenbergii, T. flammea, T. gracielae, T. hallbergii ssp. lloydi, T. huajuapanensis, T. illecebrosa, T. meleagris, T. mexicana ssp. mexicana, T. mortonii, T. multiflora and T. vanhouttei ssp. vanhouttei. The plant material is part of the germplasm collection of the Centre for Wildlife Conservation in Tenancingo, Estado de México. Fresh leaf sections were taken from each individual. All of the samples were processed in the Plant Molecular Biology Laboratory, Faculty of Agricultural Sciences of the Universidad Autónoma del Estado de México.

RAPD and ASSR analyses

DNA extraction

The genomic DNA was isolated from 100 mg of frozen leaves of each species, following a protocol with Plant DNAzol^®Reagent (Invitrogen™). The DNA was resuspended in 50 μL of TE buffer (Tris-HCl EDTA) and was stored at -20 °C until ready for use.

Amplification by PCR and electrophoresis of the DNA

Thirteen RAPD primers and five ISSR primers of type anchored were used for the PCR (Table 1).

PCR mix was performed in 10 μL containing 2 μL of 5X My Taq Reaction buffer (15 mM MgCl₂ and 15 mM dNTPs) (Bioline™), 0.1 μL My Taq Polymerase (Bioline™), 0.2 μL of the primer (20 μM) (Invitrogen™) and 1 μL of DNA (10 ng).

The amplification cycles for the primers Y24, Y37, Y38 and Y41 were: an initial cycle of 5 min at 94 °C, 1 min at 54 °C and 2 min at 72 °C; 41 cycles of 1 min at 94 °C, 1 min at 54 °C and 2 min at 72 °C followed by one final extension cycle of 1 min at 94 °C, 1 min at 54 °C and 5 min at 72 °C. For the primers P619 and P635 the cycles were: one cycle of 4 min at 94 °C; 40 cycles of 1 min at 94 °C, 3 min at 44 °C and 2 min at 72 °C followed by one cycle of 5 min at 72 °C. The cycles for the primers P628 and P647 were: one cycle of 4 min at 94 °C; 40 cycles of 1 min at 94 °C, 3 min at 50 °C and 2 min at 72 °C followed by one cycle of 5 min at 72 °C. The cycles for the primers P473 and P475 were: one cycle of 2 min at 92 °C; 40 cycles of 1 min at 91 °C, 80 sec at 50 °C and 2 min at 72 °C followed by one cycle of 10 min at 72 °C. The cycles for the primers P495, P496 and P497 were: one cycle of 2 min at 92 °C; 39 cycles of 1 min at 91 °C, 80 sec at 52 °C and 2 min at 72 °C followed by one cycle of 10 min at 72 °C.

Finally, for the primers 3'-ASSR02, 3'-ASSR15, 3'-ASSR20, 3'-ASSR29 and 3'-ASSR35 the cycles were performed according to Yamagishi et al. (2002).

The DNA fragments amplification was performed in a thermocycler Mastercycler gradient (Eppendorf^®) model Hamburg 22331-246227. The separation of the fragments was realized in horizontal electrophoresis chambers (Easycast™ ), with type II agarose gels (Sigma^®) at 1 %, to which 3 μL of ethidium bromide (Sigma^®) were added. The running conditions for each sample were 80 V and 120 mA for 80 min. The DNA bands were photographed under ultraviolet light using a UV Transilluminator BioDoc-It Imaging System (UVP™). The sizes of the amplification products were estimated with a DNA ladder (100 bp to 3000 bp) (Fermentas™).

Statistical analysis

Banding patterns were constructed considering each band as absent, (0), or present, (1). To determine genetic relationships, a dendrogram was constructed for each primer group with the data using the POPGENE program (Yeh and Boyle, 1999) with the UPGMA method based on the Nei (1972) matrix genetic distances. The total number of bands (TB), polymorphic bands (PB) and percentage of polymorphic bands (%P) were calculated using the same program.

 

RESULTS AND DISCUSSION

In the present study, PCR amplification with 10, 15 and 20 length base RAPD primers, and 17 length base ASSR led to reproducible fragment patterns for all species of Tigridia evaluated. In total, with both markers 163 bands were detected, in which 150 (92.02 %) were polymorphic. It should be noted that the 18 primers generated 27 specific bands (Table 1). Among the 15 Tigridia species evaluated, T. alpestris ssp. obtusa showed the major number of specific bands (5), whereas T. bicolor, T. illecebrosa and T. mortonii did not show any.

RAPD analysis

The RAPD primers of 10 b showed 41 total bands, in which 12 bands were specific; the average of total fragments generated per primer were 10.25 with a polymorphism of 95.12 %. For the 15 b primers, those values were 41, 5, 6.83 and 82.93 %, respectively, and for the 20 b primers, 37, 8, 12.33 and 94.59 %, respectively. In total, RAPD primers pooled (10, 15 and 20 b) showed 119 bands in which 25 were specific, and the average of total fragments generated per primer were 9.15 with a polymorphism of 90.76 % (Table 1).

Debener et al. (1996) reported the use of RAPD primers to analyze the genetic variation among wild species and cultivars of Rosa spp. and they proved their effectiveness to show a high polymorphism to differentiate the used genotypes. Likewise, Kasaian et al. (2011) used RAPD markers of 10 b for molecular characterization of five species of Juniperus L. endemic to Iran, allowing a clear distinction of each. Our results also showed that, with shorter length of the primer, the percentage of polymorphism was higher. The reason why RAPD primers of 10 b were more efficient, as compared with those of 15 or 20 b, could be due to the temperature used during the stage of alignment (54 °C for primers of 10 b, 44 °C-50 °C for the primers of 15 b, and 52 °C for primers of 20 b), because the binding of the oligonucleotides with their complementary sites is more stable when using a higher temperature (Espinosa, 2011). In contrast, Debener and Mattiesch (1998), Yamagishi et al. (2002) and Piña-Escutia et al. (2010a), while characterizing Rosa multiflora, Lilium and T. pavonia, respectively, mentioned that the primers of 20 b were the most efficient to amplify polymorphic bands, compared to other RAPD primers (10 or 15 b).

The dendrogram based on UPGMA analysis for pooled (10, 15 and 20 b) RAPD data showed the genetic differentiation of 15 wild species of Tigridia. The genetic distance (GD) among the species ranged from 0.16 (between T. illecebrosa and T. huajuapanensis) to 0.57 (between T. multiflora and T. augusta). Tigridia species were grouped in four clusters: group I clustered T. alpestris ssp. obtusa, T. augusta and T. bicolor ; group II included T. durangense, T. ehrenbergii ssp. ehrenbergii, T. gracielae, T. huajuapanensis, T. illecebrosa, T. mexicana ssp. mexicana, T. mortonii, T. multiflora and T. vanhouttei ssp. vanhouttei; group III clustered T. hallbergii ssp. lloydi and T. meleagris; and group IV included only T. meleagris (Figure 1a).

In the dendrogram, the species were not grouped according to the classification system of Molseed (1970) and did not show any correlation with phenology (data not shown). However, Wen and Hsiao (2001) suggest that the species collected in different geographical areas can be grouped within the same group, showing a correlation between the genetic diversity and geographic location of a collected species. In some cases, it was possible to establish a correlation with the altitude of geographical area of the species collected. Thus, in the group II, T. huajuapanensis and T. illecebrosa have a growth restricted to desert scrub of the states of Puebla and Oaxaca (Mexico), between 1500 and 1900 m amsl (Cruden, 1968; 1975; Espejo-Serna and López-Ferrari, 1996). Furthermore, in group IV T. meleagris was left apart from the rest of the species probably because is the most rampant specie of Tigridia in Mexico, ranging from 1300 to 2000 m amsl (Espejo-Serna and López-Ferrari, 1996). This was also possible because T. meleagris is the only specie belonging to the subgenus Hydrotaenia: meleagris complex (nectarials structures shaped tip) (Molseed, 1970).

ASSR analysis

The total number of reproducible bands with five ISSR primers anchored-type was 44, with an interval of 300 to 2000 bp in the size of the amplified fragments. Of the 44 bands, 42 were polymorphic, with an average of 8.8 bands per primer. Polymorphic bands ranged between 71.4 to 100 %, with an average of 95.45 % per primer (Table 1).

These results proved the high efficiency of anchored microsatellite (ASSR) to assess the genetic diversity of 15 species of the genus Tigridia, because four of the five primers used showed 100 % of polymorphism. The effectiveness of the ASSR primers as well as the type of the motive replicate is possibly favoured by the sequence of its anchor. The CT motive sequences produce higher polymorphism with respect to the AT replicates (Pradeep et al., 2002; Hu et al., 2003), which despite being the most abundant in the plant genomes, have the disadvantage that the amplification of DNA fragments is low; this may be due to the semi-complementarily of the primer in the alienation stage of the PCR (Fang and Roose, 1997).

With the five primers, two specific bands were generated in two of the 15 species tested, of which the species T. augusta and T. ehrenbergii ssp. ehrenbergii showed one band each, and these bands were amplified by the 3'ASSR20 and 3'ASSR02 primers. Furthermore, all ASSR primers used showed polymorphic banding patterns, and it was possible that one, the 3'ASSR02, could differentiate the 15 species of Tigridia Jussieu (Figure 2 and Table 1). These results are similar to those reported by Piña-Escutia et al. (2010b) who found that, with usinly only one ASSR primer, was enough to molecularly characterize nine varieties of T. pavonia. Those markers showed genetic diversity among varieties, distinguishing each one of them. In addition, Bautista-Puga et al. (2011) evaluated four botanical varieties of Sprekelia formosissima (L.) Herbert using ASSR primers ranging 57 to 100 % polymorphism among those varieties, generating a molecular profile for an unambiguous identification for each of them.

The dendrogram generated from ASSR data (Figure 1b) formed three groups among species of Tigridia: group I included T. alpestris ssp. obtusa, T. augusta, T. huajuapanensis, T. multiflora, T. vanhouttei ssp. vanhouttei, T. flammea, T. illecebrosa, T. mexicana ssp. mexicana and T. Mortonii; group II included T. bicolor, T. durangense, T. ehrenbergii ssp. ehrenbergii and T. gracielae; and group III included T. hallbergii ssp. lloydi and T. meleagris. The dendrogram did not show any correlation between the morphological characteristics and phenology of these species (data not shown), but the same phenomenon occurred within the species of restricted distribution as in the dendrogram of RAPD pooled data. The highest genetic association (G_D = 0.89) was found between T. mexicana ssp. mexicana and T. durangense, whereas the least related were T. vanhouttei spp. vanhouttei and T. multiflora (G_D = 0.14).

Finally the dendrogram with pooled data (RAPD and ASSR) (Figure 1c) showed a range of G_D of 0.18 (between T. vanhouteii ssp. vanhouttei and T. multiflora) to 0.6 (between T. meleagris and T. hallbergii ssp. lloydi). Their topology had the same characteristics of the RAPD pooled dendrogram.

By the use of RAPD and ISSR anchored primers, this research showed a wide genetic diversity among 15 species of Tigridia, which might be explained by the fact that Mexico is the center of origin and dispersal of the genus (Rodríguez and Ortiz-Catedral, 2003a, 2003b). Similar results were reported in populations of Passiflora edulis Sims using RAPD markers in Brazil, where the studies show high levels of diversity in this species, explained by the fact that this country is center of origin and diversity of the species (Bellon et al., 2007).

 

CONCLUSIONS

The analysis showed the effectiveness of 10, 15 or 20 bases Random Amplified Polymorphic DNA primers and 17 bases Inter Simple Sequence Repeat of type Anchored in the detection of polymorphism and genetic differentiation of 15 wild species of Tigridia. The study revealed that 10 base random primers and 17 base anchored primers were more efficient to detect polymorphism and genetic differentiation of the 15 Tigridia species.

 

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