Belg. J. Bot. 142 (2) : 204-208 (2009) © 2009 Royal Botanical Society of Belgium
Short note
IDENTIFICATION AND CHARACTERIZATION OF EIGHT NUCLEAR MICROSATELLITE LOCI IN THE GLASSWORT GENUS SALICORNIA (AMARANTHACEAE) A. VANDERPOORTEN1,*, O. RASPÉ2, A. M. RISTERRUCCI3, L. GOHY1 and O. J. HARDY4 1
Research associate of the Belgian Fund for Scientific Research (FNRS) at Université de Liège, Institut de Botanique, B22 Sart Tilman, B-4000 Liège, Belgium 2 National Botanic Garden of Belgium, Domein van Bouchout, B-1860 Meise, Belgium 3 UMR 1098 CIRAD, TA A96/03 Avenue Agropolis, 34398 Montpellier Cedex 5, France 4 Research associate of the Belgian Fund for Scientific Research at Université Libre de Bruxelles, Faculté des Sciences, Service Evolution Biologique & Ecologie, CP 160/12,50 Av F. Roosevelt, B-1050 Brussels, Belgium (* Author for correspondence; e-mail:
[email protected]) Received 6 February 2009; accepted 10 November 2009.
ABSTRACT. — Eight nuclear microsatellite loci were identified using the method of microsatellite-enriched libraries in the glasswort genus Salicornia. These markers yield specific alleles for discriminating diploid and tetraploid lineages and thus provide a reliable and efficient tool for routine determination of ploidy level in the genus. Within both the diploid and tetraploid lineages, the microsatellites further display a clear biogeographic signal. A significant partitioning of genetic differentiation was found between the diploid populations (Fst = 0.31; p < 0.001). The new microsatellite markers described here thus offer the appropriate amount of variation to address issues of species delimitation and biogeographic history in the genus. KEY WORDS. — Salicornia, microsatellite, principal component analysis, species delimitation.
INTRODUCTION Because of the low levels of molecular variation in widely used DNA regions such as large, non-coding cpDNA or ITS, species-level patterns and processes in plants are still globally understudied (BAKKER et al. 2005). Microsatellites, which typically exhibit high rates of mutation, therefore increasingly appear as promising tools for the identification of individuals, breeds, cultivars and species, especially in groups of organisms like
bryophytes (KORPELAINEN et al. 2008, SHAW et al. 2008), or other land plants with reduced morphologies (VON STACKELBERG et al. 2006). Within angiosperms, the glasswort genus Salicornia is a case-in-point for exhibiting strikingly low levels of molecular and morphological variation. Morphologically, Salicornia species are characterized by a strongly convergent, succulent morphology adapted to a harsh, semi-aquatic and salty environment. They exhibit a combination of extremely reduced characters, such as almost
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absent leaves and naked flowers, and a high level of plasticity in global habit and color, offering minimal and often misleading taxonomic information. As a result, even the identification of diploids and tetraploids, which represents the first dichotomy in the classification of the genus, can be problematic. Tetraploids tend to be larger and have flowers subequal in size, whereas diploids tend to be smaller and have a large, central flower surrounded by two smaller flowers per inflorescence. Numerous intermediate occur, however, and species delimitation within the diploid and tetraploid lineages often proves to be extremely challenging (KADEREIT et al. 2007). The increasing interest for molecular markers in such groups is thus evident. However, non-coding nuclear regions such as ITS and ETS, which proved extremely variable in other groups, only revealed weakly differentiated lineages (SHEPHERD et al. 2004, KADEREIT et al. 2007, MURAKEÖZY et al. 2007, KALIGARIC et al. 2008). For example, these gene sequences do not allow for clear species delineation between S. pusilla and S. europaea s.l.
(MURAKEÖZY et al. 2007), yet perhaps the most morphologically distinct diploid species. One reason for the lack of resolution provided by the noncoding nuclear ribosomal regions is that the genus is of fairly recent origin and underwent fast range expansions (KADEREIT et al. 2007). In this note, we define and characterize eight specific nuclear microsatellite loci in Salicornia. MATERIAL AND METHODS Genomic DNA was isolated from ca. 100 mg shoot using the cetyltrimethylammonium bromide (CTAB) procedure outlined by DOYLE & DOYLE (1987), without RNase treatment. The microsatellite loci were identified using the method of microsatellite-enriched libraries following the protocol described in HUTSEMÉKERS et al. (2008) from DNA of a single population of S. patula (Marsal, northeastern France), a diploid species. One hundred clones were sequenced. The obtained sequences were compared against each other to identify overlaps among amplified products. Redundant sequences were discarded and 25 primer pairs were designed using
Table 1. Primer sequences and characterization of eight nuclear microsatellite loci in Salicornia for a sample of both diploid and tetraploid specimens from Mediterranean and Atlantic populations. Locus GenBank accession number S1 FJ479621 S2
FJ479622
S5
FJ479623
S7
FJ479624
S8
FJ479625
S9
FJ479626
S10
FJ479627
S19
FJ479628
Primer sequences 5’-3’ (Tm) F: TCCCTTTTATTTGGTTTTCA (54.9°C) R: AAAACGGTTCATTCTCTTTG (54.5°C) F: AGCAACCATTTCAGAGCTT (55.1°C) R: TCTTACATCCCGCAACAT (54.8°C) F: TGGACCAATAACGAAGCTA (54.3°C) R: TTCTTCCCTTCAATGTTCA (54.0°C) F: TTTGGTGAAGTGATAAGCAA (54.4°C) R: GACCTCAGAGCTGCATTT (53.7°C) F: GGAGCATCTAGGCTTCCA (56.4°C) R: GCTTGCTACCACATTTTGC (56.9°C) F: AGGTGGGAAGAAGAGCAA (55.8°C) R: CCCCAAACTTTAATTTCAGC (56.4°C) F: TCGCCTATATGGACTGGT (53.8°C) R: TGCTAACTTTGGGTGGTT (54.0°C) F: AGCTTCCACTAGCAAACAAG (54.9°C) R: ACCCTATTTATGTCATTGATCC (54.7°C)
Repeat array
Number of alleles Alle size (diploids/tetraploids) range
(TG)12 5 (5/2)
124-135
(TG)9
2 (1/2)
122-124
(CA)8
4 (2/2)
102-110
(AC)8
6 (5/6)
121-129
(TG)9
4 (3/4)
122-128
(TG)9
2 (2/2)
103-111
(AG)14 5 (4/5)
166-174
(GT)8
175-183
Note. F = forward primer; R = reverse primer; Tm = melting temperature.
3 (2/2)
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3 (ROZEN & SKALETSKY 2000). PCR amplifications were performed in 12 µL, with 2 µL DNA, 0.5 µL of each primer (10 µM, HEX or FAM-labelled forward and unlabelled reverse primers), 1.25 µL of each dNTP (2.5 mM), 3 µL of 25 mM MgCl2, 1.25 µL of buffer 10×, and 0.5 U of Taq polymerase (Roche). Thermocycling consisted in a denaturation step of 5 min at 94°C; 35 cycles of 45 s 94°C, 45 s at 50°C and 90 s at 72°C; and finally an elongation step of 7 min at 72°C. The designed primers were also tested for crossamplification of the microsatellite loci, and the use of these loci to discriminate among morphologically wellcharacterized diploids and tetraploids on the one hand, and among species or populations at the same level of ploidy on the other, was assessed by analyzing a sample of Corsican and Atlantic populations. The Atlantic sample, which originates from the Authie estuary (northwestern France), consists of 19 diploid specimens of S. ramosissima and 11 tetraploid specimens of S. fragilis. The Corsican sample includes 15 diploid specimens of S. patula and 9 tetraploid specimens of S. emerici from several localities along the eastern coast of the island. The obtained data were analyzed by Principal Component Analysis (PCA) on a complete disjunctive table of presence/absence of alleles for each individual. PRIMER
RESULTS AND DISCUSSION From those 25 initial primer pairs, eight were eventually retained for their easy and consistent amplification and polymorphism. Although the library was initially built on a specific population of a diploid species, the identified loci could be successfully amplified in other diploid and tetraploid species. As in other cases of successful crossspecies amplification from species-specific primers (e.g., SPOON & KESSELI 2008, TAKAGI et al. 2008), the success in the cross-species amplification in Salicornia probably results from the close phylogenetic affinities among the tested species, as evidenced by the extremely low levels of sequence variation in noncoding chloroplast and ribosomal DNA (SHEPHERD et al. 2004, KADEREIT et al. 2007, MURAKEÖZY et al. 2007). Details on the eight pairs of primers are provided in Table 1. Along the PCA axis 1, which accounts for 30.3% of the total allelic variance, diploid and tetraploid individuals are clearly separated (Fig. 1). The tetraploids are indeed consistently character-
Fig. 1. PCA of the complete disjunctive table of presence/absence of alleles at eight microsatellite loci for a sample of diploid and tetraploid Salicornia from the Atlantic coast in northern France and the Mediterranean coast in Corsica. PCA1 (horizontal axis) and PCA2 (vertical axis) account for 30.3% and 12.1% of the total allelic variance, respectively. Values of the highest correlation coefficients between the axes and the alleles are given at the extremity of each axis.
MICROSATELLITE LOCI IN SALICORNIA
ized by fixed heterozygosity at loci S2 and S19 and in one population at S5, which is consistent with a disomic inheritance of alleles and an allopolyploid origin. By contrast, all the diploids investigated are homozygous at these loci. Tetraploids further differ from diploids by specific alleles at loci S2 (allele size 124), S5 (allele size 102 and 104), and S19 (allele size 183), while exhibiting a higher frequency of allele 125 at locus S7 than diploids. Although examination of a larger number of specimens is required, the preliminary results presented here suggest that these markers have a good potential to determine the ploidy level of any morphologically ill-characterized plant or any herbarium specimen. Within tetraploids, the Atlantic lineage (S. fragilis) differs from the Mediterranean lineage (S. emerici) by its homozygosity at locus S5 (allele size 104), whereas the Atlantic plants were consistently found to be heterozygous at that locus (allele size 102+104). Within diploids, The Atlantic plants (S. ramosissima) are characterized by the frequent occurrence of a specific allele at locus S19 (allele size 175), while the Corsican plants (S. patula) most often share a specific allele at locus S10 (allele size 172). In fact, a significant genetic differentiation was found between the two diploid populations (Fst = 0.31; p < 0.001 after 1000 individual permutations). These results suggest that the new microsatellite markers described here offer the appropriate amount of variation to address issues of species delimitation and biogeographic history in Salicornia. ACKNOWLEDGEMENTS This research was performed thanks to a grant (2.4504.05) from the Belgian Fundamental and Collective Research Funds (FRFC).
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TAKAGI M., SAKAI K. & TANIGUCHI N., 2008. — Isolation and characterization of 13 microsatellite markers for the viviparous surfperch Ditrema temmincki (Embiotocidae) and crossspecies amplification. Mol. Ecol. Resour. 8: 1030-1033.
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