Genetic Diversity and Transferability of Rubus Microsatellite Markers to South American Rubus Species

Rubus L. (Rosaceae) is grown extensively worldwide, in altitudes ranging from 0 to 4500 m above sea level. Found in six continents, this genus is reported to contain approximately 800 species due to biological processes such as hybridization, apomixis, and polyploidy that weaken species boundaries (Thompson, 1995). Rubus has been divided into 12 subgenera of which only a few species have been domesticated (Table 1).


Introduction
Rubus L. (Rosaceae) is grown extensively worldwide, in altitudes ranging from 0 to 4500 m above sea level.Found in six continents, this genus is reported to contain approximately 800 species due to biological processes such as hybridization, apomixis, and polyploidy that weaken species boundaries (Thompson, 1995).Rubus has been divided into 12 subgenera of which only a few species have been domesticated (Table 1).
This species presents traits of two subgenera-Idaeobatus and Rubus-possibly being a fertile amphidiploid or allotetraploid (n=7, 4x=28) (Delgado et al., 2010;Thompson, 1997).Sympatrically with R. glaucus, some other wild Rubus species are found in the Andean cordillera along with the introduced and cultivated Eurasian R. idaeus L. Because hybridization is a common process that affects species of any genus (Randell et al., 2004), it is reasonable to believe that gene flow is currently taking place between species of this genus.
Studies on the genetic diversity of Rubus have been carried out in temperate species, such as Rubus idaeus (Graham & Mcnicol, 1995;Graham et al., 1997;Parent & Fortin, 1993) and Rubus occidentalis (Parent & Page, 1998), and Asian species (Amsellem et al., 2000).These works used RAPD, RFLP, and SCAR markers as well as SSR (Antonius-Klemola, 1999).ITS are also been used to study hybrids of R. idaeus and R. caesius (Alice et al., 1997) .These markers made it possible to confirm the genetic origin of the hybrids and further phylogenetic and evolutionary studies in Rubus (Alice, 2002).Recently, major advances have been achieved worldwide in the use of molecular markers in temperate species of Rubus, such as DNA fingerprinting to study and characterize genotypes, development of linkage maps, marker-assisted selection, and mapping of QTLs (Antonius-Klemola, 1999;Graham et al., 2002).
To date, molecular markers such as randomly amplified polymorphic DNA (RAPD), amplified fragment length polymorphisms (AFLP) (Marulanda et al., 2007), and microsatellites (simple sequence repeats, SSRs) (Marulanda et al., 2011) have been used to study the genetic diversity of the Andean blackberry.Previous work carried out by Marulanda et al. (2007) and Marulanda & López (2009) on the genetic diversity of Colombian blackberries identified high phenotypic and molecular plasticity in the R. glaucus species known as the "Castilla" blackberry in Colombia's central Andean area.Other wild Rubus species present in the Andean region are found near farms where the "Castilla" blackberry is commercially grown.These plants were also submitted to morphological, agronomic, and molecular characterizations using AFLP and SSR molecular markers (Marulanda & López, 2009).A genomic library enriched for microsatellite sequences was recently developed for R. glaucus.
This chapter presents the results of the molecular characterization of wild and cultivated Rubus species collected in the central Andes of Colombia using SSR markers from other Rubus species available in Genbank, together with 11 microsatellite markers isolated from R. glaucus and characterized in 39 samples of Rubus (Table 2).Intra-and inter-specific differences between R. glaucus and its wild relatives were established, generating not only information on the current status of populations, their uses, and distribution, but also information considered crucial to launch a breeding program for R. glaucus.

Plant material and DNA extraction
A total of 39 Rubus samples were collected at altitudes ranging from 1800 to 2455 m above sea level in the central Andes of Colombia (between 1° 42´10.7 ´´ and 6° 99¨44´´N and 72° 92´80´´ and 76° 25´ 35.9´´W), and placed on silica gel (1:10, plant tissue: silica gel) (Table 2).DNA was isolated using the Plant DNAeasy Miniprep kit (QIAGEN ® ), following the manufacturer's instructions.Several samples did not show any DNA after the isolation procedure so it was necessary to reprocess these samples following the Doyle & Doyle (1990) protocol.In all cases, samples were purified using the protocol described by Castillo (2006).

Analysis with SSR markers
A total of 36 microsatellite sequences from other Rubus species, R. idaeus (23 primer pairs) (Series RhM, RiM and Rubus) and R. occidentalis (2 primer pairs) (Series mRaCIRRI), and 11 microsatellites from R. glaucus were used (Table 3).The microsatellite named as "Rg" was developed using a genomic library enriched for microsatellite sequences from a cultivated genotype of R. glaucus, following the protocol described by Billotte et al. (1999).

Statistical analysis
The allelic diversity of the SSR was evaluated by determining polymorphism information content (PIC) value, as described by Bonstein et al. (1980) and cited and modified by Anderson et al. (1993), as described in Equation 1: Where P ij is the frequency of the jth pattern, i is the sum, and n are the patterns.To measure the utility of the marker systems, average heterozygosity and expected and observed heterozygosity were calculated.The partitioning of genetic variation within and among the groups by the SSR marker system was achieved by analysis of molecular variance (AMOVA) using the same software.Analyses were performed in GenAlex (Peakall & Smouse, 2006) and Arlequin v.3.5 (Excoffier & Lischer, 2010).

Genetic diversity and variability
A total of 41 loci and 133 alleles were detected.The number of alleles observed for each locus ranged from 2 to 6, with an average of 4.6 alleles per locus.The PIC value varied between 0.07 and 0.61 (average 0.48), and the discriminating power (D) ranged from 0.05 to 0.52 (average 0.27).Observed heterozygosities (Ho) were 0.078-1.0(average 0.84) and expected heterozygosities (He) were 0.07-0.582(average 0.473).The highest PIC value (0.61) was found in the Rg-D7 locus, which presented a high number of alleles (5).To compare the efficiency of markers to identify varieties, the D value of each primer was estimated.The highest D value (0.52) was also found in the Rg-D7 locus (Table 3).
Similar results were reported by Castillo (2006), who used 12 SSRs to analyze an extensive collection of North American Rubus (raspberry) germplasm.Results indicated from 3 to 16 alleles per locus, with an average of eight alleles per locus and a total number of alleles of 96.More recently, Flores et al. (2010) isolated 12 microsatellites from SSR-enriched genomic libraries of R. idaeus.
Another measure of genetic variability is the presence of exclusive alleles per loci and genotype (Tables 4 and 5).The genotypes presenting the highest number of exclusive alleles are listed in Table 4, with R. idaeus genotype CVM11 ranking highest, which could be attributed to the fact that most of the SSRs used are derived from R. idaeus.Of the evaluated microsatellites, those of the series "Rubus" were one of the most polymorphic groups and detected the highest number of alleles in the study population.It should be mentioned that exclusive alleles also appear in wild genotypes and in genotypes 106 and 107, which belong to the species R. urticifolius.The loci in which the private alleles were detected are very important for genotype differentiation, particularly in the case of the thornless genotypes.Fig. 2 presents the results of the principal coordinates analysis.There is no clear differentiation of genotypes based on collection sites; however, the genotypes belonging to the species R. urticifolius (37, 44, 106, and 107)  1 R. alceifolius (Amsellem et al., 2001); 2 R. idaeus (Graham et al., 2002(Graham et al., , 2004) ; 3 R. idaeus and blackberry "Marion" (Marulanda et al., 2010 (data not published).* PIC = polymorphic information content; A = allele number; D = discrimination power; He = expected heterozy heterozygosity.In addition to the genetic diversity measurements already mentioned, the analysis of molecular variation (AMOVA) revealed 98%variability among all genotypes and 2% variation between populations (Michalakis & Excoffier, 1996) (Table 6).These data agree with those observed in the principal coordinates analysis, where variation is mostly attributed to individuals variation.Kollmann et al. (2000) concluded that genetic variability in Rubus is determined by the plant propagation system and demonstrated that there is an effect of cross-pollination between polyploid Rubus species.This type of crossbreeding influences seed and fruit quality positively, while increasing ploidy levels and taxonomic proximity.www.intechopen.com

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The Molecular Basis of Plant Genetic Diversity 160

Transferability of microsatellite sequences
Microsatellites RhM and RiM developed from R. idaeus showed cross-species amplification in R. glaucus genotypes and in other two wild species of Rubus, with 2 and 3 alleles per locus.High D values were found for loci RhM018 (0.5094) and RiM015 (0.4451).The "Rubus" microsatellite markers, also from R. idaeus, were the most polymorphic and presented the highest number of alleles per locus: Rubus 105b with 6 alleles and Rubus 107a with 5.The microsatellite markers from R. alceifolius showed 3 alleles and D values ranging from 0.23 to 0.2566.The R. glaucus microsatellites (Rg series) amplified for other wild species, with the number of alleles ranging from 2 to 5 per locus.
The cross-species amplification data suggested that microsatellites developed for R. idaeus, R. alceifolius, and R. glaucus can be potentially useful for genetic diversity studies of different Rubus species.In the case of conservation programs, they should prove useful for characterizing natural populations and germplasm collections, as well as for determining the degree of relatedness between individuals or groups of accessions.
The microsatellites developed by Amsellem et al. (2001) to study R. alceifolius, subgenus Malachobatus, that grows in Southeast Asia were used for the characterization of Andean Rubus, the transferability and applicability of microsatellites of R. alceifolius to study and evaluate the diversity of Rubus species in the American Andes were demonstrated; results were similar to those obtained in Asian species.Amsellem et al. (2001) also observed amplification from 3 to 4 alleles per individual in the species R. alceifolius, confirming the suspicion that this is a tetraploid species.Based on the analysis carried out by Amsellem et al. (2001), the present study produced between 3 and 5 alleles for R. glaucus and between 2 and 4 alleles for R. urticifolius., suggesting that both cultivated and wild materials of R. glaucus have ploidy levels greater than those of R. urticifolius.This polyploidy was also described by Hall (1990), who explained that Rubus species used in plant breeding programs have produced euploid and aneuploid hybrids and that diploid, triploid, tetraploid, hexaploid, septaploid, octaploid, and nonaploid cultivars have been selected, most of them tetraploids.

Conclusion
Wild forms are also usually found at sites where Rubus species are cultivated, particularly in forest clearings, along roadsides, and on hillsides.Both cultivated and wild forms have the potential for interacting in different ways with cultivated materials.Cultivars can influence the genetic diversity of natural populations through gene transfer by pollen and wild populations are a potential source of genetic material for improvement programs.
This evaluation of the status of genetic resources of the species R. glaucus and related wild species serves to provide guidelines for conservation and breeding efforts aiming to promote the development of cultivated species important for the rural economies of South America's Andean region.
Using microsatellites from other Rubus species has proven to be a very useful strategy to differentiate between wild and cultivated R. glaucus genotypes, as well as between thorny and thornless cultivars.
The development of a genomic library enriched with microsatellites and the design of microsatellite sequences for the Andean specie Rubus glucus, is allowing a deeper comprehension of the genetic variability existing among cultivated and wild genotypes as well as the relationships between the cultivated specie and the wild relatives.
The Analysis of molecular variation (AMOVA) showed a higher variability distributed between genotypes than between populations, which agrees with the results obtained in the principal coordinates analysis.

Table 2 .
Samples of Rubus species, accessions of R. glaucus, and collection sites.
sec, 65 °C (−1 °C/cycle) for 30 sec and 72 °C for 1 min; 35 cycles of 94 °C for 15 sec, annealing temperature (°C) for 30 sec and 72 °C for 1 min; and 72 °C for 5 min.Genetic Diversity and Transferability of Rubus Microsatellite Markers to South American Rubus Species 155 are separate from the species R. glaucus, both cultivated and wild.Likewise, the species R. idaeus is separate from the species R. urticifolius

Table 3 .
Microsatellite sequences and characteristics of each SSR used to evaluate Rubus materials.and wild genotypes of R. glaucus.Genotype CVM Wild of the species R. glaucus is notably separate from the genotypes of the same species.

Table 4 .
Summary of private alleles.

Table 5 .
List of samples with one or more private alleles.

Pr i nci pal Coor di nat es
Fig. 2. Principal coordinates analysis among Rubus genotypes based on genetic distance .