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Polymorphic Microsatellite DNA Markers in Opuntia spp. Collections I. Chessa, P. Erre, M. Barbato and G. Nieddu Department of Economics and Woody Plant Ecosystems University of Sassari Via De Nicola 9, 07100 Sassari Italy

J. OchoaFacultad de Agronomia y Agroindustrias Universidad Nacional de Santiago del Estero Argentina

Keywords: SSRs, cactus pear, genetic variability, germplasm Abstract

In the present study, five SSRs from a novel set of microsatellite loci and 8 previously isolated are analyzed in different species and cultivars of Opuntia, selected from two ‘on field’ collections located in Italy and Argentina. The objectives were to investigate the genetic variability in the cactus pear and assess the ability of the tested SSR markers in identifying and discriminating Opuntia accessions at species and intra-species levels. All SSRs produced polymorphic amplifications in the 29 accessions analyzed. The mean value of the polymorphic index content (PIC) for the SSR loci provided good discriminating ability for the assessment of genetic diversity in the genotypes included in the study. The diversity indices were higher in the species population than in the cultivars populations, but the cultivated accessions retained more diversity than expected for a domesticated species.

INTRODUCTION

The Mediterranean area is one of the important zones of cactus pear introduction and diffusion. Commercial plantations have been established in Italy mainly directed toward fruit production and based on few cultivars and numerous local biotypes, belonging to the Opuntia ficus-indica and O. amyclaea species. The systematic studies conducted in Italy, aimed at protecting the genetic resources of Opuntia from the Mediterranean area, have given origin to the collection set up at the University of Sassari (Chessa, 2010).

In most of the arid and semi-arid zones of Argentina cactus pear grown by smallholders is part of the traditional farmsetting (Caloggero and Parera, 2004). More recently the plant cultivation successfully developed and a number of plantations from medium to large size took place as a promise for higher rewards (Ochoa, 2005). Cropping mainly relies on different ecotypes of local cultivars and on cultivars introduced from other cactus pear producing countries. A wider variability than that found in the Mediterranean areas has developed in Argentina, linked also to the occurrence of many Cactaceae and wild Opuntia species. The genetic resources of Opuntia and other Cactaceae have been collected and characterized for the morphological traits at the Universidad Nacional de Santiago del Estero in Argentina (Ochoa, 2003).

However, germplasm characterization based on molecular traits provides more reliable information, and has attained special attention due to its increased use in crop improvement and the selection of desirable genotypes for breeding crops. Molecular fingerprinting, using RAPDs and ISSR, has been applied to the management of cactus pear collections (Wang et al., 1998; Garcia-Zambrano et al., 2006; Zoghlami et al., 2007; Chessa et al., 2008; Luna-Paez et al., 2007) and to elucidate the hybrid origin of Opuntia species (Griffith, 2004). The genetic relationships among different species and the variability of collected genetic resources were investigated through AFLP (Labra et al., 2003; Garcia-Zambrano et al., 2009). The codominant nature of SSRs has made them the marker of choice to unravel cases of erroneous species designation (Helsen et al., 2009) and to investigate the differentiation level among cactus pear genotypes of different origin (Caruso et al., 2010).

Knowledge of genetic diversity and of relationship among species and cultivars in

Proc. 7th International Congress on Cactus Pear and CochinealEds.: A. Nefzaoui et al. Acta Hort. 995, ISHS 2013

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the cactus pear is useful to provide documentation on collections of genetic resources, and to remove the uncertainties in the accessions classification, both wild and cultivated. In the present paper we aimed at evaluating the degree of polymorphism in a novel set of microsatellite loci, isolated by the University of Sassari in different species and cultivars of Opuntia (Erre et al., 2011). The microsatellites previously identified by Hensen et al. (2007) in the O. echios cultivars (var. echios and var. gigantea) were added for evaluation and fingerprinting purpose. Others objectives were to identify and characterize genetic diversity at species and intra-species level in Opuntia, as well as provide information on the current state of Opuntia genetic resource conservation in the germplasm collections from Italy and Argentina. MATERIALS AND METHODS Plant Material and SSR Markers

The genomic DNA was isolated from young cladodes of 29 Opuntia species and cultivars (Table 1). The accessions examined were located at two fields ex situ collections, one hosted by the University of Sassari in Italy and the other one hosted by the Universidad Nacional de Santiago del Estero in Argentina. Five SSRs from a novel set of 10 microsatellites developed within a pool of Opuntia DNA samples (Erre et al., 2011) were analyzed in this study and compared with eight SSRs from those previously isolated by Helsen et al. (2007).

Microsatellite amplification was conducted with the following conditions: each 20 μl reaction mixture contained 10-20 ng genomic DNA, 8 µl Hot Master Mix 2.5X (5-PRIME) and 0.2 μm of each primer. Polymerase chain reaction (PCR) amplifications started with one denaturation cycle at 95°C for 15 min, followed by 35 cycles at 95°C for 30 s, 56°C for 30 s, 72°C for 30 s and a final extension step at 72°C for 10 min. Forward primers of the 13 primer pairs were fluorescently labelled with 6-FAM (blue). A short run electrophoresis in agarose gel was performed to ensure amplification occurred. Fragment analysis was performed on an ABI prism 3100 Genetic Analyzer (PE Applied Biosystems) and ROX400 was used an internal size standard. Data Analysis

Allele calling and sizing were performed using the genescan 3.1 (Applied Biosystems) and the genotyper 2.5 software. Due to the polyploid nature of the species studied, partial heterozygosity makes it impossible to score genotypes exactly. We have scored co-dominant microsatellites as dominant markers with alleles as presence-absence data using POPGENE 1.31 software (Yeh and Boyle, 1997). Features of the microsatellite markers and their corresponding PCR primers are summarized in Table 2. For the diversity estimation of 13 SSR loci and to evaluate the genetic diversity within species and cultivars population, percentage of polymorphic loci (P%), observed mean number of alleles per locus (no), effective mean number of alleles per locus (ne), Shannon’s information index (Is) and Nei’s gene diversity (He) were calculated at population level.

The coefficient of genetic similarity of the investigated accessions was calculated using the following equation: SMab=m/(m+n), where m is the number of bands that are shared by genotypes ‘a’ and ‘b’ and n is the number of bands that are polymorphic in the genotypes ‘a’ and ‘b’. Cluster analysis was performed on the basis of the matrix of genetic similarity coefficients. The unweighted pair group method of arithmetic means (UPGMA) was used, employing for this purpose NTSYS-PC version 2.1 (Rohlf, 1998). Results of the performed grouping are presented in the form of a dendrogram. RESULTS AND DISCUSSION

All SSRs produced polymorphic amplifications in the 29 accessions analysed. A total of 279 alleles were observed with an average of 21.46 at locus. Genotyping results enabled to identify 94 private alleles for Opuntia species and 14 alleles specific of the germplasm from Argentina. This result reflects diversity between the cultivar and species.

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Polymorphism varied considerably, specific alleles per locus ranged between 8 and 34 (Table 2). The mean value (0.72) of the polymorphic index content (PIC) for the SSR loci provided good discriminating ability for the assessment of genetic diversity in the genotypes included in the study. The average PIC values for each primer pair indicates that the Opufic primers are generally more discriminant, with the SSR loci Opufic13 (0.941), Opufic03 and Opufic14 (0.849) loci as the most informative.

Concerning the genetic diversity within groups of Opuntia spp, the percentage of polymorphism (P%) at the population level is high both in the species group (93.48%) and in the cultivars population (65.22%). Shannon’s information index (Is) at the population level is 0.302 for the species group and 0.294 for the cultivars group, with the average value of 0.298 (Table 3). Nei’s gene diversity (H) is 0.192 in the cultivars group and 0.178 for the species group, with an average value of 0.185. As expected, the standard diversity parameters were higher in the species population than in the cultivars populations. Selection process is likely to result in significant reductions of genetic variation, as has been observed in most domesticated plant species (Doebley, 1992). However, the cultivated accessions retained more diversity than expected for domesticated species. This event could be justified by the contribution of cultivars from different regions and by the limited artificial selection and crop breeding in cactus pear.

The cluster analysis identified the genetic relationship between species and cultivar genotypes and demonstrated the potential and the reliability of microsatellite markers for genome analysis (Fig. 1). The genetic tree divides into two clusters at similarity value of 0.64. The not classified accession named ‘Rossa Armerina’ clustered separately into one independent branch, indicating its assignment to a cactus pear related species. The first group contained all the wild Opuntia spp. analyzed, the Nopalea cochenellifera, but also the cultivated genotypes Opuntia ficus indica ‘1281’ and Opuntia ficus indica ‘Achefri’. The dissimilarity of the accession classified as Opuntia ficus indica ‘1281’ with the O. ficus indica species has been suggested by Wang et al. (1998), based also on cladode and plant morphology.

The Opuntia species included in this group shared high similarity values, and no geographical clustering was observed among them. The second group included all the cultivated genotypes and other Opuntia species, strictly related to O. ficus-indica, such as the O. megacantha and O. streptacantha. The genetic similarity between O. ficus-indica and O. megacantha was revealed by AFLP and cpSSR (Labra et al., 2003), while Griffith (2004) by means of Bayesian phylogenetic analyses of nrITS DNA sequences evidenced a close relatedness of O. ficus-indica with a group of species including O. megacantha and O. streptacantha. The polyphyletic origin of the cactus pear, domesticated from ancestral stock of arborescent, fleshy-fruited plants is also supported by the microsatellite analysis conducted by Caruso et al. (2010). CONCLUSIONS

The level of polymorphism and the relatively high number of alleles detected suggest that these markers can be used for both inter and intra-specific studies. The developed markers proved to enable the analysis of different species using the same SSR loci set. The information obtained can shed some light on the classification of Opuntia species, based on their allelic profiles.

Estimates of genetic diversity and the relationships between germplasm collections from different regions are very important to identify genetically diverse, agronomically superior accessions for the improvement of cactus pear. The SSRs data combined with agronomic, qualitative, morphological, and phenological data will create a useful instrument to facilitate the management and use of cactus pear collections. However, it should be noted that due to the presence of polyploidy within the Opuntia genus, the SSR may have a limited capacity to represent true genetic distances between cultivars, owing to the difficulty of identifying the allelic profile at locus.

A better understanding of the effectiveness of the different molecular markers is considered a priority step toward management of cactus pear collection and a prerequisite

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for more effective breeding program. Literature Cited Caloggero, S. and Parera, C.A. 2004. Assessment of prickly pear (Opuntia ficus-indica)

varieties and their possible planting systems. Spanish Journal of Agricultural Research 2(3):401-407.

Chessa, I. 2010. Cactus pear genetic resources conservation, evaluation and uses. FAO Cactusnet Newsletter, Special Issue 12:43-53.

Chessa, I., Nieddu, G., Nieddu, M. and Erre, P. 2008. Variabilità inter e intra-specifica in Opuntia spp. nel Mediterraneo. Atti VIII Convegno Nazionale sulla Biodiversità: 143-145.

Caruso, M., Currò, S., Las Casas, G., La Malfa, S. and Gentile, A. 2010. Microsatellite markers help to assess genetic diversity among Opuntia ficus indica cultivated genotypes and their relation with related species. Plant Syst Evol. DOI 10.1007/ s00606-010-0351-9.

Doebley, J.F. 1992. Molecular systematics and crop evolution. p.202-222. In: D. Soltis, P. Soltis and J. Doyle (eds.), Molecular Systematics of Plants. Chapman-Hall, New York.

Erre, P., Nieddu , G. and Chessa, I. 2011. Identification of microsatellite loci in Opuntia spp. and their characterization in cultivars and species. Acta Hort. 918:327-332.

Garcia-Zambrano, E.A., Salinas, G., Vazquez, R., Cardenas, E. and Gutierrez, A. 2006. Clasificacion y estimacion de la diversidad genetica de Nopal Opuntia spp en base a descriptores fenotipicos y marcadores genetico moleculares. ΦYTON, International Journal of Experimental Botany 75:125-135.

Garcıa-Zambrano, E.A., Zavala-Garcıa, F., Gutierrez-Diez, A., Ojeda-Zacarıas, M.C. and Cerda-Hurtado, I. 2009. Estimation of the genetic diversity of Opuntia spp. using molecular markers AFLP. ΦYTON, International Journal of Experimental Botany 78:117-120.

Griffith, M.P. 2004. The origins of an important cactus crop, Opuntia ficus-indica (Cactaceae): new molecular evidence. Am. J. Bot. 91:1915-1921.

Helsen, P., Verdyck, P., Tye, A. et al. 2007. Isolation and characterization of polymorphic microsatellite markers in Galapagos prickly pear (Opuntia) cactus species. Mol. Ecol. Notes 7:454-456.

Helsen, P., Verdyck, P., Tye, A. and Van Dongen, S. 2009. Low levels of genetic differentiation between Opuntia echios varieties on Santa Cruz (Galapagos). Plant Syst. Evol. 279:1-10.

Labra, M., Grassi, F., Bardini, M., Imazio, S., Guiggi, A., Citterio, S., Banfi, E. and Sgorbati, S. 2003. Relationships in Opuntia Mill. Genus (Cactaceae) detected by molecular marker. Plant Sci. 165:1129-1136.

Luna-Paez, A., Valadez-Moctezuma, E., Barrientos-Priego, A.F. and Gallegos-Vázquez, C. 2007. Characterization of Opuntia spp. by means of seed with RAPD and ISSR markers and its possible use for differentiation. J. PACD 9:43-59.

Nei, M. 1973. Analysis of gene diversity in subdivided populations. Proc. Natl. Acad. Sci. USA 70:3321-3323. doi: 10.1073/pnas.70.12.3321.

Ochoa, J.M. 1994. El cultivo de la tuna en Argentina. I Jornadas Regionales de Frutas No Tradicionales, San Juan, Argentina, May 21-23. 7p.

Ochoa, J.M. 2003. Cactus pear (Opuntia spp.) varieties main characteristics at Republica Argentina. FAO Cactusnet Newsletter, Nùmero especial:3-29.

Ochoa, J.M. 2005. Manejo de los tunales hacia un sistema de aprovechamiento integral. FAO Cactusnet Newsletter, Nùmero especial 10:64-72.

Rohlf, F.J. 1998. NTSYS: numerical taxonomy and multivariate analysis system. Sneath, P.H.A. and Sokal, R.R. 1973. Numerical Taxonomy: The Principles and Practice

of Numerical Classification. San Francisco, Freeman. Wang, X., Felker, P., Burrow, M.D. and Paterson, A.H. 1998. Comparison of RAPD

marker patterns to morphological and physiological data in the classification of

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Opuntia accessions. Journal of the Professional Association for Cactus Development 3:3-14.

Yeh, F.C. and Boyle, T.J.B. 1997. POPGENE, Version 1.1. Department of Renewable Resources, University of Alberta, Edmonton, Alberta.

Zoghlami, N., Chrita, I., Bouamama, B., Gargouri, M., Zemni, H., Ghorbel, A. and Mliki, A. 2007. Molecular based assessment of genetic diversity within Barbary fig (Opuntia ficus indica (L.) Mill.) in Tunisia. Sci. Hort. 113:134-141.

Tables Table 1. Analyzed genotypes and accessions. Accessions Location ProvenanceO. robusta Italy SicilyO. stricta Italy Sardina (private collection) O. dillenii Italy Sardina (private collection) O. lindheimeri Italy Sardina (private collection) O. polyacantha Italy Sardina (private collection) O. rastrera Italy Sardina (private collection) O. soherensii Italy Sardina (private collection) O. sulphurea Italy Sardina (private collection) O. amyclaea (Bianca Bronte) Italy SicilyNopalea cochenillifera Italy USAGialla lungomare (not identified) Italy SicilyO. ficus-indica Achefri Italy MoroccoO. ficus-indica (BSC) Italy SiciliRossa armerina (not identified) Italy SiciliyO. basilaris Italy Sardina (private collection) O. megacantha Argentina MexicoO. crassa verde Argentina MexicoO. crassa rosa Argentina MexicoO. ellisiana Argentina USA (New Mexico) O. streptacantha Argentina North AfricaO. matudae (xoconostle) Argentina MexicoO. ficus-indica 1294 Argentina USA (Texas)O. ficus-indica 1282 Argentina USA (Texas)O. ficus-indica 7 Argentina ArgentinaO. ficus-indica verde Argentina ArgentinaO. ficus-indica 11 Argentina ArgentinaO. ficus-indica Roja Argentina ArgentinaO. ficus-indica 1281 Argentina USA (Texas)O. ficus-indica 1321 Argentina USA (Texas)

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Table 2. Characteristics of polymorphic microsatellite loci.

Locus Repeat

type

Size range (bp)

No. of alleles

Alleles/ ind.

Average PIC

values

Erre et al., 2011

Opufic01 (CT)16 148-184 19 1-12 0.765 Opufic03 (TG)12 134-163 14 2-8 0.849 Opufic04 (TG)12 196-222 17 2-9 0.746 Opufic13 (TC)12(AC)11 136-190 31 2-18 0.941 Opufic14 (CTT)7…(CTT)10 143-282 43 3-21 0.849

Helsen et al., 2007

Opuntia2 (AG)14(CG)4 201-217 9 1-4 0.438 Opuntia4 (GA)12 98-124 14 1-3 0.656 Opuntia8 (CT)5(TC)12GC(TC)5 102-137 19 1-8 0.713 Opuntia9 (AG)15 146-173 27 1-7 0.803 Opuntia10 (CT)9 159-191 8 2-5 0.372 Opuntia12 (TC)4C(TC)12 226-272 21 1-12 0.696 Opuntia13 (AG)12 238-262 19 1-8 0.742 Opuntia21 (TC)14 93-166 28 1-11 0.814

Table 3. Measures of genetic diversity within groups of Opuntia spp. Groups P% no ne Is H Cultivars 65,220 1.652 1.321 0.294 0.192 Species 93,480 1.930 1.251 0.302 0.178 Mean 79,350 1.791 1.286 0.298 0.185 S.D. 0.197 0.049 0.006 0.010 P%: percentage of polymorphism; no: observed number of alleles per locus; ne: effective number of alleles per locus; Is: Shannon’s information index; H: Nei’s gene diversity.

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Figures

Fig. 1. UPGMA dendrogram describing genetic similarity of the examined accessions.

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