Microsatellite variation in potato landraces from the island of La Palma

Nineteen microsatellite markers were used to fingerprint a set of 19 potato landraces from the island of La Palma (Canary Islands). These landraces represent relicts of early introductions from South America, although most are commonly cultivated by local farmers. The SSR primers detected 62 polymorphisms, 13 of which were present in all landraces. Several accessionand group-specific markers were detected. Jaccard similarity coefficients were estimated from the molecular data and UPGMA cluster analysis was performed. Some cultivars with related common names clustered together. In some cases, e.g., for the ‘Conejera’ landrace, the molecular patterns were discrepant with previous species assignments, suggesting the need for a more detailed morphological and comparative study of these accessions. Additional key words: characterization, genetic diversity, germplasm, Solanum tuberosum, SSR markers.


Introduction
During colonial times, the Canary Islands were the first point of entry into Europe of goods from South America, including novel plant species such as the potato.Modern potato cultivars are very different to this original material, but in the Canary Islands a number of Andean introductions have been maintained and cultivated since the 16th century.Local cultivars can there-fore be found that are very similar to the native material brought from Peru (Zubeldia et al., 1955).These cultivars provide lucrative crops for local growers since, despite their low yields, they demand high prices.
The first references to the presence of potatoes in the Canary Islands date from 1567 (for Grand Canary Island) and 1574 (for Tenerife) (Hawkes and Francisco-Ortega, 1993).In 1868, Alvarez-Rixo published the first inventory of Tenerife's potatoes.Zubeldia et al. (1955) assigned the traditional cultivars of the Canary Islands to the following taxa: Solanum tuberosum subsp.andigena, S. tuberosum subsp.tuberosum, and S. chaucha
(formerly S. mamilliferum).Solanum chaucha is a triploid hybrid between the tetraploid S. tuberosum subsp.andigena and the diploid S. stenotonum (Zubeldia, 1955).Marrero (1992) and Gil et al. (2000) determined the geographical distribution of the traditionally grown cultivars in the Canary Islands and evaluated their agronomic performance.Based on these studies, Rios (2002) collected accessions from Tenerife in an attempt to identify duplicates and classify the varieties.This author published a detailed morphological and ecophysiological characterization of the island's cultivars.On the island of La Palma, similar work is being carried out by the Cabildo Insular de La Palma (the La Palma local government).Researchers have collected 19 accessions from two places on the island for their classif ication.All of these accessions are thought to be morphologically different (Lorenzo R., pers.comm.), and share similar common names.On La Palma, homonyms are commonly related to the place of cultivation and the agronomic characteristics of the growing area.Molecular markers are useful in cultivar identification, the analysis of biodiversity (Ritter et al., 2005), and for examining the phylogenetic relationships related to autoploidy and amphidiploidy in potato (Martinez-Zapater and Oliver, 1984).Microsatellite markers (SSR) are particularly helpful since they are highly polymorphic, represent co-dominant markers, and are generally well-conserved across related species.Moreover they are simple to use in PCR (Powell et al., 1996).The f irst SSR study of potato was based on DNA sequences from public databases (Veilleux et al., 1995).SSR markers have subsequently been used to study genetic relationships among S. tuberosum cultivars (Provan et al., 1996;Schneider and Douches, 1997).Milbourne et al. (1998) published a set of 112 potato SSRs located on all 12 chromosomes of the genome.Ashkenazi et al. (2001) used SSRs to study phylogenetic distances among wild and cultivated potato species.Raker and Spooner (2002) were able to distinguish S. tuberosum subsp.tuberosum from S. tuberosum subsp.andigena using SSR markers.Ghislain et al. (2004) used 22 SSR markers to analyse over 900 native potato accessions from the Andes belonging to eight different Solanum species, and finally Barandalla et al. (2006) used them to classify 41 local potato cultivars from 10 locations on Tenerife.
The aim of the present study was to use SSR markers to analyse and compare the molecular relationships among potato landraces from the island of La Palma.

Material and Methods
Molecular analyses were performed on 19 potato landraces collected on the island of La Palma (28º40'N/ 17º37'W; 2426 m maximum altitude) by the Cabildo Insular de La Palma, which have yet to be fully morphologically characterised.The commercial variety 'Kennebec' (S. tuberosum) and the Tenerifian accessions 'Peluca Blanca' (S. tuberosum subsp.tuberosum), 'Bonita Colorada' (S. tuberosum subsp.andigena) and 'Yema de Huevo' (S. chaucha) were used as references.
Table 1 shows the accession codes, common names, places of origin and the current taxonomic assignment of each entry.Based on their common names, groups of local varieties such as 'Marcialas' and 'Corraleras' were distinguished.Other landraces such as 'Moruna', 'Malgara', 'Morada', 'Colorada' and 'De año' represented individual entries.
DNA extraction was undertaken using the DNAeasy Plant Mini Kit (Qiagen, Valencia, CA, USA).Concentrations were determined by electrophoresis in 0.7% agarose gels and comparison to a 1kb DNA ladder standard (Invitrogen, Carlsbad, CA, USA).Nineteen of the 22 SSR primers used by Ghislain et al. (2004) were employed in the present work (Table 2).One of the primers of each SSR was modified with a 5'-end M13 extension (Steffens et al., 1993).A complementary M13 primer labelled with the fluorescent infrared dye IRD800 (LI-COR, Lincoln, Nebraska, USA) was added to each PCR reaction.This helped reduced the number of labelled primers.PCR reactions were performed in a 10 µl volume containing 1 µl of 10 × PCR buffer, 200 µM of dNTPs, 2.5 mM of MgCl 2 , 0.2 µM of each primer (forward and reverse), 0.18 µM of M13 IRD800 primer (MWG, Genotec, Spain), 0.5 units of Taq polymerase (LINUS, Teknovas, Spain), and 30 ng of genomic DNA.PCR was carried out in a Robocycler Gradient 96 thermocycler (Stratagene, La Jolla, CA, USA) using the following cycling profile: 2 cycles of 1 min at 94ºC, 2 min at 63ºC and 35 s at 70ºC, 18 touchdown cycles of 45 s at 94ºC, 45 s at 62ºC (-1ºC each 2 cycles), and 45 s at 70ºC, 20 cycles of 30 s at 92ºC, 30 s at 53ºC, and 1 min at 70ºC, plus a final elongation step of 5 min at 72ºC.Amplification products were separated on 6% denaturing polyacrylamide gels.SSR fragments were detected using a LI-COR 4200-S1 DNA Sequencer (LI-COR Biosciences, Eversberg, Germany), and fragment analysis performed following the manufacturer's instructions (LI-COR, 1997).
Each band or fragment on the gels was scored for presence or absence.These data were used to calculate similarity coefficients between the 19 accessions.A cophenetic matrix was produced from the tree matrix and compared to the distance matrix generated by the Jaccard coefficients (Jaccard, 1901).Cluster analyses were performed using the UPGMA method (Rohlf, 2001) and employing NTSYS-PC software (Rohlf, 2001; supplied by Exeter Software, Setauket, NY, USA).The polymorphism index content (PIC), a measure of allelic diversity, was calculated according to Nei's coefficient (Nei, 1973), PIC = 1-Σ(p i 2 ), where p i is the frequency of the i th polymorphism detected in the germplasm.The polymorphism frequency distribution was analysed using the PROC UNIVARIATE procedure of the SAS program (SAS, 2000).

Results
Table 2 shows the polymorphisms detected by each SSR.The landraces showed a total of 62 SSR frag-ments, ranging from one (monomorphic) to six (polymorphic) per SSR.Only 13 of them were present in all materials; the other 49 showed a varying degree of polymorphism among accessions.PIC values ranged from 0 to 0.78; STM0030 had a high PIC value and a maximum of five markers.
Figure 1 shows the dendrogram obtained by UPGMA clustering using the amplification results for the 19 SSRs.The cophenetic matrix derived from the cluster analyses was in good agreement with the original similarity matrix (r 2 = 0.956).Setting the cut-off point for the average similarity coefficient at 0.78 distinguished seven groups.The first was formed by 'Moruna', 'Jaragana', 'Colorada' and the Tenerif ian cultivar of S. tuberosum subsp.tuberosum 'Peluca Blanca'.All the accessions of this group belong to S. tuberosum subsp.tuberosum.The second group included four accessions from La Palma ('De año', 'Corralera Blanca', 'Corralera

Discussion
The dendrogram of the cluster analysis shows that the landraces from La Palma are all different.In some cases they have similar names -but this is because they share the place or method of cultivation.For example, the group of 'Corraleras' is formed by four landraces (PS-LP3, PS-LP6, PS-LP13 and PS-LP15), all of them different, yet they share a similar name because they are commonly cultivated in vineyards.The accessions from La Palma were recently collected and morphological studies on them are as yet incomplete.Accessions PS-LP7, PS-LP9 and PS-LP12 were introduced to the island in the 20th century and are not traditionally cultivated (Lorenzo R., pers.comm.).The accessions PS-LP9 and PS-LP12 are both 'Conejeras' but are different molecularly and morphologically.Traditional names often give ethnobotanical and ethnoagricultural information, which is useful in conserving genetic plant resources, but this work helps to clarify the relationship and origin of some of the accessions from La Palma, and suggests they should be renamed.
In the dendrogram (Fig. 1), group VI (the 'Marcialas') represents S. tuberosum subsp.tuberosum germplasm.The members of this group are morphologically very different from those of other groups, with more stems, few flowers and berries, and bigger leaves (Rios, 2002).This group includes five different landraces: PS-LP2,

Figure 1 .
Figure 1.Potato landraces from La Palma: dendrogram of the cluster analysis based on the Jaccard similarity coefficient, using the UPGMA clustering method.

Table 1 .
Number of SSR polymorphisms in 19 accessions of potato landraces from La Palma, three cultivars from Tenerife, and a commercial cultivar of S. tuberosum (cv.Kennebec)

Table 2 .
Polymorphisms detected by SSR markers in 19 potato landraces from La Palma Missing data means some totals may not reach 19. 2 PIC: polymorphism information contents.

Table 3 .
Accession-and group-specific SSR polymorphisms detected in potato landraces from La Palma