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Revista de la Facultad de Ciencias Agrarias. Universidad Nacional de Cuyo

versión impresa ISSN 1853-8665versión On-line ISSN 1853-8665

Rev. Fac. Cienc. Agrar., Univ. Nac. Cuyo vol.50 no.2 Mendoza dic. 2018

 

ORIGINAL ARTICLE

Analysis of genetic variability in vitro regenerated buffelgrass plants through ISSR molecular markers

Análisis de variabilidad genética en plantas regeneradas in vitro de buffelgrass mediante marcadores moleculares ISSR

Edgardo Carloni 1, Eliana López Colomba 1, Andrea Ribotta 1, Mariana Quiroga 1, Exequiel Tommasino 1, Sabrina Griffa 1, Karina Grunberg 1, 2

1 Instituto de Fisiología y Recursos Genéticos Vegetales (IFRGV), CIAP-INTA. Av. 11 de Septiembre 4755. C. P. X5020ICA. Córdoba, Argentina. carloni.edgardo@inta.gob.ar; edgardocarloni@gmail.com

2 Consejo Nacional Investigaciones Científicas y Técnicas (CONICET), Godoy Cruz 2290, C1425FQB, CABA, Argentina.

Originales: Recepción: 06/10/2016 - Aceptación: 24/08/2017


ABSTRACT

Genetic variability can be generated through in vitro culture via somaclonal variation. This tool can be potentially useful in a breeding program involving apomictic buffelgrass genotypes. The aim of this work was to evaluate inter simple sequence repeats (ISSR) as molecular markers to detect genetic variation in in vitro buffelgrass regenerated plants. Six plants regenerated from in vitro anther culture, via somatic embryogenesis were used, as well as the anther donor genotype (RN 51) as control. Of a total of 26 ISSR primers tested, 22 amplified, detecting 12% polymorphism with a divergence between 5 and 24% from RN 51. Amplification products were observed with the primers containing di-, tri- or tetra-nucleotide sequences, with or without additional nucleotides at the 3′ end. The most informative primers were those containing the repetitive sequences GACAn, AGn or GAn. Moreover, the regenerants transplanted at field conditions differed in morphological characteristics among them and with respect to RN 51. This study confirms that ISSR are useful to identify genetic variability in in vitro regenerated buffelgrass plants.

Keywords: Apomixis; Somatic embryogenesis; Somaclonal variation; Anther culture; ISSR; Genetic diversity

RESUMEN

El cultivo in vitro permite generar variabilidad genética mediante variación somaclonal. Este fenómeno puede ser potencialmente útil en un programa de mejora en genotipos apomícticos de buffelgrass. El objetivo del presente estudio fue evaluar inter secuencias simples repetidas (ISSR) como marcadores moleculares en la detección de variación genética en plantas regeneradas in vitro de buffelgrass. Se utilizaron seis plantas regeneradas mediante el cultivo in vitro de anteras, vía embriogénesis somática, y el genotipo dador de anteras (NR 51) como control. De un total de 26 cebadores ISSR probados, 22 de ellos amplificaron, detectando un 12% de polimorfismo con una divergencia del 5 al 24% con respecto al material NR 51. Se observaron productos de amplificación con los cebadores que contienen secuencias di-, tri- o tetra-nucleótidos, con o sin nucleótidos adicionales en el extremo 3′. Los cebadores más informativos fueron aquellos que contenían secuencias repetitivas GACAn, AGn o GAn. Conjuntamente, las plantas trasplantadas a campo manifestaron características morfológicas diferentes entre ellas y con respecto al NR 51. El presente estudio confirma que el empleo de ISSR permite identificar variabilidad genética en plantas regeneradas in vitro de buffelgrass.

Palabras claves: Apomixis; Embriogénesis somática; Variación somaclonal; Cultivo de anteras; ISSR; Diversidad genética


INTRODUCTION

In vitro cell or tissue culture offers several advantages to plant species. The use of this biotechnological tool has resulted in regenerated plants showing spontaneous genetic variability with respect to the donor plant. This phenomenon is known as somaclonal variation (20) and may be undesirable in some in vitro propagated species (9). In apomictic cultivars of buffelgrass [Pennisetum ciliare (L.) Link syn. Cenchrus ciliaris L.], however, those changes can be potentially useful as a source of genetic variability that can be beneficial in breeding programs (23) of this important forage grass worldwide in arid and semiarid regions (4, 18).

Genetic variation reported in several in vitro regenerated species include phenotypic changes (33), alterations in chromosome number or specific regions (19), point mutations (11, 24), and activation of transposable genetic elements (21), among others. Accordingly, all these factors should be considered when identify and analyze the occurrence of a somaclone (20). Morphological characters may be one of the most practical, cost-effective and easy methods to evaluate somaclonal variation (33). Nevertheless, these studies in in vitro regenerated plants usually require a comprehensive approach, including several nuclear DNA analysis techniques (2, 7).

In buffelgrass, measurement of nuclear DNA content by flow cytometry (FCM) has been suggested as an alternative and plays an important role in the analysis of micropropagated plant suffering changes in ploidy levels (8). Also randomly amplified polymorphic DNA (RAPD) are mentioned to identify somaclones from induced mutations of embryogenic callus treated with ethylmethane sulfonate (EMS) and in vitro selection (23).

In others species, one of the approaches used in those studies is ISSR (inter simple sequence repeat) molecular markers (10, 12, 26, 30). This technique is based on polymerase chain reaction (PCR) and uses a single primer complementary to the microsatellites (28). One variant to this method consists of adding nucleotides in the 3' end of the primer, known as anchored primer (34). Each amplified band is a locus and corresponds to the DNA sequence located between two similar or inverted microsatellites. This technique has the advantage that primer design does not require previous knowledge of the DNA sequence to be amplified (5, 16). There is scarce information about the use of this type of molecular markers in buffelgrass, except for studies that determined genetic diversity in wild populations (1, 17). Here we aimed to evaluate inter simple sequence repeats (ISSR) as molecular markers to detect genetic variation in in vitro buffelgrass regenerated plants.

MATERIALS AND METHODS

Plant material

The study included six plants of the R1 generation (plants obtained by in vitro anther culture, via somatic embryogenesis) which showed genetic instability in nuclear DNA content by flow cytometry (8) and anther donor plant registration number 51 accession (RN 51). All the plants were transplanted at field in the Instituto de Fisiología y Recursos Genéticos Vegetales of the Instituto Nacional de Tecnología Agropecuaria (INTA) (Córdoba, Argentina) for further evaluation.

Inter simple sequence repeats (ISSR)

Genomic DNA of each regenerated and donor plant was isolated from frozen leaves (-20°C) using the Nucleon PhytoPure (GE Healthcare) commercial kit and following the manufacturer's instructions. Pure DNA was resuspended in 50 μl of distilled water. DNA concentration in each sample was quantified by absorbance reading (A260/280 nm) in a NanoDrop ND-1000 V3.5 spectrophotometer (NanoDrop Technologies, USA).

A total of 26 ISSR universal primers were tested; they were obtained from sequences published on line the "UBC Primer Set #9" (31) and from previous works in other plant species (14, 26, 32) (table 1, page 4).

Table 1. Analysis of genetic variability of in vitro regenerated plants buffelgrass by ISSR.

Tabla 1. Análisis de la variabilidad genética en plantas regeneradas in vitro de buffelgrass mediante ISSR.

The amplification reaction was performed in a final volume of 20 μl, containing 2 μl DNA (25 ng/μl), 1X buffer solution (Tris-HCl 6 mM pH 8.8, KCl 50 mM), 0.8 μl MgCl2 (25 mM), 4 μl of each dNTP (1 mM), 4 μl of the primer (5 μM) and 0.2 U of GoTaq® DNA polymerase (Promega, USA). Amplification consisted of following steps: 94°C for 3 min, 35 cycles at 93°C for 30 s, 55°C for 1 min, 72°C for 1 min 30 s, followed by a final extension of 72°C for 5 min. To improve the band patterns of those primers that failed to amplify, different annealing temperatures were tested (14) and in some reactions, 0.5 μl (10 mg/ml) of bovine serum albumin (BSA) was added. A minimum of three reactions per primer were performed to increase resolution of the band pattern. Amplifications were conducted in an Eppendorf Mastercycler 5333 thermocycler.

Amplification products were run for two hours at 100 V in 2% agarose gels in 0.5 X TBE buffer solution. A sample volume of 12 μl was loaded per lane [10 μl amplification product + 2 μl 5X loading buffer] as well as a GeneRuler 100-pb molecular weight marker, Plus DNA Ladder (Thermo Scientific Inc) as reference marker to calculate the size of the bands obtained with the ISSR.

PCR products were staining with ethidium bromide (10 mg/ml, GIBCO BRL) and fragments were observed under UV light (312 nm). Finally, the gel images were captured using BIORAD, Molecular Imager® Gel DocTM XR System (USA).

Phenotypic evaluation

To identify the ocurrence and observe phenotypic changes, RN 51 used as control and in vitro regenerated plants were transferred to an experimental plot in a completely randomized design. Thus, plant materials were transplanted at 1 m distance between them and morphological characters of leaf, stem and panicle were measured (25).

Phenotypic tests were performed during two crop seasons (2013/14 and 2014/15).

Statistical Analyses

The molecular profiles obtained using ISSR were coded by performing a matrix of binary data per primer and genotypes, where "1" and "0" indicated presence and absence of bands, respectively, and each band was regarded as a locus. Genetic relationships between individuals were evaluated using the Jaccard's similarity index [J=a/(a+b+c)] (15, 26, 27), where a, b and c are absolute frequencies for events (1.1), (1.0) and (0.1), respectively. To obtain a matrix of genetic distances between individuals a transformation of the Jaccard’s index (sqrt (1-S)) was performe d. From the obtained data the proportion of polymorphic loci (PMF (95)) and polymorphism information content (PIC) were calculated. The PMF was calculated as the number of polymorphic loci / total number of loci, while PIC was estimated using the following equation:

where:

pi = the frequency of the

i = allele

n = the number of alleles (6)

The distance matrix was subjected to a principal coordinate analysis (PCoA) and to unweighted pair group method with arithmetic mean (UPGMA) hierarchical clustering. All the analyses conducted with the binary data matrix were performed using Info-Gen statistical software (3).

To confirm phenotypic variability in the in vitro regenerated materials, morphological variables of R1 plants were compared with those of the anther-donor plant (RN 51) using t test. To determine the degree of grouping of R1 and RN 51 material, a clustering analysis was performed using the average linking clustering UPGMA by applying the Euclidean distance as proximity measure. The analyses were performed using InfoStat statistical software (13).

RESULTS

Inter simple sequence repeats (ISSR)

The ISSR generated amplification products in the DNA samples extracted from buffelgrass plants. Of a total of 26 primers tested, 22 amplified clear and reproducible bands. Table 1 (page 4) shows a descriptive analysis of amplification data obtained with ISSR primers. A total of 460 bands were generated, of which 58 were polymorphic (12%). Fifteen (68%) polymorphic primers were detected, with a proportion from 4 to 40% of polymorphic bands. The primers RAF16 (0.32), ISSR8A (0.24), UBC840A (0.24) and ISSR1A (0.23) produced the highest polymorphism information content. Of them, ISSR1A and UBC840A had the highest proportion of polymorphic bands (0.38 and 0.34, respectively).

Most of the polymorphic ISSR (RAF4, UBC834, ISSR8A, 17899, UBC827, UBC840A, ISSR1A, UBC816, ISSR4) generated the two types of expected data (absence and presence of alleles).

However, in comparison to donor plant, some primers only showed absence of alleles (AE2, UBC864, ISSR1B and RAF16), whereas ISSR9B showed presence of new alleles.

The amplification profiles generated with ISSRs 17899, RAF4, UBC827 and UBC834 are displayed as an example in figure 1.

Figure 1. Detection of genetic variability using ISSR in vitro regenerated buffelgrass plants. Agarose gel electrophoresis (2 %) of the PCR product obtained with the following ISSR: (A) 17899, (B) RAF4, (C) UBC827, and (D) UBC834.

Figura 1. Detección de variabilidad genética utilizando ISSR en plantas regeneradas in vitro de buffelgrass. Electroforesis en gel de agarosa al 2 % del producto de PCR obtenido con los siguientes ISSR: (A) 17899, (B) RAF4, (C) UBC827 y (D) UBC834.

To understand the degree of similarity of molecular profiles between the in vitro regenerated individuals (R1) and RN 51, the 22 ISSR that amplified were used for diversity studies and distance analyses.

The results obtained from the sqrt (1-S) transformation of Jaccard’s similarity index indicates that the distance coefficients vary between 0.05 and 0.24 (5 and 24%, respectively), between in vitro regenerated plants and control (table 2).

Table 2. Distance matrix of six in vitro regenerated buffelgrass plants and genotype RN 51 (C), obtained from the transformation sqrt (1-S) of the Jaccard's similarity index based on 22 ISSR primers.

Tabla 2. Matriz de distancia de seis plantas regeneradas in vitro de buffelgrass y del genotipo NR 51 (C), obtenida de la transformación sqrt (1-S) del índice de similitud de Jaccard basado en 22 cebadores ISSR.

The analysis including only the coefficients of the in vitro regenerated plants showed values ranging between 0.11 and 0.29 (11 and 29%, respectively), indicating not only divergence from RN 51 but also differences between in vitro regenerated plants.

The results of the principal coordinate analysis (PCoA) show that the most important principal components (PCs) (PC 1 and PC 2) (figure 2, page 8), which account for almost 62% of the total variability, shows two groups; one of them is composed of a single genotype (R1-t31), which showed the greatest differences among the in vitro regenerated materials in PC 1.

Figure 2. Biplot of six in vitro regenerated buffelgrass plants (R1-t4, R1-t5, R1-t6, R1-t31, R1-t32 and R1-t33) and RN 51 genotype used as control (C), obtained from sqrt (1-S) transformation of Jaccard’s similarity index based on 22 ISSR primers.

Figura 2. Biplot de seis plantas regeneradas in vitro de buffelgrass (R1-t4, R1-t5, R1-t6, R1-t31, R1-t32 y R1-t33) y del genotipo NR 51 utilizado como control (C), obtenido de la transformación sqrt (1-S) del índice de similitud de Jaccard basado en 22 cebadores ISSR.

In turn, PC 2, differentiates two groups, one of them including the material used as anther donor or control (C) and the other including two regenerants that were very different from the rest (R1-t32 and R1-t33).

Figure 3 (page 8) showed the results of the dendrogram generated by UPGMA clustering method.

Figure 3. Dendrogram of six in vitro regenerated buffelgrass plants (R1-t4, R1-t5, R1-t6, R1-t31, R1-t32 and R1-t33) and RN 51 genotype used as control (C), obtained via UPGMA method through the analysis of ISSR markers over a distance matrix calculated from the sqrt (1-S) transformation of the Jaccard's similarity index.

Figura 3. Dendrograma de seis plantas regeneradas in vitro de buffelgrass (R1-t4, R1-t5, R1-t6, R1-t31, R1-t32 y R1-t33) y del genotipo NR 51 utilizado como control (C), obtenido por el método UPGMA mediante el análisis de marcadores ISSR, sobre una matriz de distancia calculada de la transformación sqrt (1-S) del índice de similitud de Jaccard.

The former cophenetic analysis yielded a correlation coefficient of 0.97. These results further highlight the differences compared with PCoA, because if considering a distance of 0.13 as an arbitrary cut-off value (50% of the maximum distance), R1-t32 and R1-t33 individuals not formed a group and the R1-t4 individual was separated from the group that included RN 51 (C).

Phenotype evaluation

Of the six plants that were transplanted to the field (R1), five exhibited very different phenotypes from RN 51, and the remaining one did not survive. Plants R1-t5 and R1-t6, whose DNA content increased (6), showed significant differences (P < 0.05) in four morphological variables: plant height (PH), internode length (INTL), stem width (SW) and number of branches (NBR) (table 3, page 9).

Table 3. Characters of external morpholoogy of leaf, stem and panicle analyzed in 5 in vitro regenerated buffelgrass plants.

Tabla 3. Caracteres de morfología externa de hoja, tallo y panoja analizados en 5 plantas regeneradas in vitro de buffelgrass.

The lower INTL, without modifying the number of nodes (NN), gives these plants a stunt appearance (lower PH). Moreover, the plants have a lower number of branches (NBR). The material R1-t5 exhibits scarce panicles and the other one (R1-t6) has a lower panicle length (PanL) (P < 0.05). Both regenerants produce few seeds (data not shown). R1-t6 also presented a decrease in flag leaf length (FLL), flag leaf width (FLW) and flag leaf sheath length (FSL) (P < 0.05).

The plants exhibiting a reduction in DNA content (R1-t4, R1-t32 and R1-t33) (8) also showed a statistically different increase in phenotypic traits with respect to control (P < 0.05) (table 3). All the plants exhibited a decrease in PH. Plant R1-t4 had reduced FLW (thinner leaves), as well as PanL, INTL and SW. Plant R1-t32, also had smaller panicles, and smaller length of exerted peduncle (EPL) and INTL. Plant R1-t4 exhibited particularly lightercolored foliage.

Because R1-t33 and R1-t5 plants had few panicles with immature inflorescences, PanL, TEPL, EPL and flowers/branch (FL/ BR) variables and characters of leaf (FLL, FLW, FSL) were not considered in the analysis of clusters. The results of this analysis show that in vitro regenerated plants are phenotypically different from genotype RN 51. The dendrogram shows that the group including regenerants R1-32, R1-t4 and R1-t33 are the most similar to material RN 51, and that R1-t5 and R1-t6 are the most different materials (figure 4, page 10).

Figure 4. Dendrogram constructed using UPGMA method through the analysis of morphological markers, with Euclidean distance being applied as proximity measure.

Figura 4. Dendrograma construido utilizando el método UPGMA mediante el análisis de marcadores morfológicos, aplicando la distancia Euclídea como medida de proximidad.

DISCUSSION

Different molecular profiles were detected in in vitro regenerated buffelgrass plants using ISSR molecular markers, suggesting some degree of variability (16, 17, 34). There is scarce information available on the use of this type of markers in somaclonal variation in buffelgrass.

Studies conducted with ISSR in other in vitro propagated species reported very different polymorphic levels. For example, a 50% polymorphism level was detected in variants of Camellia sinensis L. (30); lower levels, of 5 and 3.9%, were recorded in micro-propagations of Musa spp. and Dictyospermum ovalifolium, respectively (10, 26). Here, total polymorphic loci were found to be 12%, which is similar to results reported for Codonopsis lanceolata (15.7%) or in clones of Camellia spp. (12.8%) (12, 15).

Likewise, the generated polymorphism showed a diferentiation degree of 5 to 24% with respect to the "control" pattern, as observed in somaclones of Zea mays L. (24). In addition, it is important to note that using the same type of molecular marker similar levels of polymorphism were obtained in wild populations of buffelgrass (1).

Amplification products were observed with the primers containing di-, tri- or tetra-nucleotide sequences, with or without additional nucleotides at the 3′ end. While most of the ISSR produced bands using the method implemented here, some of them failed to amplify. These primers were then tested at lower annealing temperatures, since molecular patterns were found to be highly modified by this factor (14), but some of these ISSR also failed to amplify. Lower temperatures could be used (16) or other additional factors should be considered, which were not studied in this work (5, 34).

The most informative primers were those containing repetitive sequences GACAn, AGn or GAn, with the addition of one or three nucleotides at the 3′end. Similar results were described in a study conducted to determine genetic diversity in wild bufflegrass populations (17).

Hence, this results confirm that the motifs (GA)n and anchors at the 3′end can be included in the ISSR markers used for different studies of this species.

The polymorphism detected by ISSR in in vitro regenerated buffelgrass plants may be attributed to genetic changes in a restricted portion of the genome, because microsatellites and their continuous bases are the hybridization site of the marker primer (16, 34). This portion of the genome usually undergoes changes during the DNA replication-repair process (22, 29), and some authors suggest that the generated polymorphism may be due to deletions or insertions that occur at the primer recognition site (24). Regardless of the type of mutations occurring during in vitro plant regeneration, they can generate gain or loss of alleles (28), as shown in this work.

Finally, plants transplanted to the field exhibited very different morphological characteristics among them and from the genetic material used as control (RN 51), thereby confirming changes in the phenotype.

This study, along with a previous one (8), clearly reveal that a group of in vitro regenerated plants undergo genetic and morphological changes with respect to the anther donor material, suggesting that in vitro culture technique is a source of somaclonal variation (20).

These observations suggest that, besides the changes detected by flow cytometry at the nuclear DNA content levels (8), in vitro regenerated plants might also have undergone specific mutations in the genome (24). It is possible that these changes restricted to a few nucleotides cannot be detected using flow cytometry. For this reason, ISSR molecular markers should be included to explore genetic variations in in vitro regenerated buffelgrass plants.

CONCLUSIONS

The use of ISSR allowed to identify genetic variability in in vitro regenerated buffelgrass plants.

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ACKNOWLEDGMENTS

We thank I. Teich for assistance with statistical analyses. This research was supported by the projects INTA-AEFP-PE No. 1126072 and CONICET (PIP 112 201101 0031).

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