Eurasian watermilfoil (Myriophyllum spicatum L.) is a perennial submerged macrophyte native to Europe, Asia, and northern Africa (Couch and Nelson, 1985). In North America, M. spicatum has been recognized as a noxious invasive plant mainly due to the rapid spread of this species (Reed, 1977; Jacono and Richerson, 2003). Myriophyllum spicatum is hexaploid, and the chromosome number (2n = 6x = 42) was reported for plants from Europe and North America (Löve, 1961; Aiken et al., 1979), whereas there was no report about the polyploid types of M. spicatum because its chromosomes were found to be too small to disclose morphological characteristics for karyotype analysis (Aiken, 1981). There have been numerous studies concerned with the ecology and management of M. spicatum, and only a few studies have revealed DNA sequence variation among different individuals (e.g., Moody and Les, 2007). No investigation has been carried out to examine genetic variation in M. spicatum at the population level; the evolutionary processes of this species are more likely distinctive due to its occurrence in exclusively aquatic habitats (Barrett et al., 1993). Therefore, we isolated 20 microsatellite markers from M. spicatum for use in investigations of genetic variation, population genetic structure, and evolutionary history of this cosmopolitan submerged species.
METHODS AND RESULTS
Total genomic DNA was extracted from the dried leaves of one individual of M. spicatum sampled from the Tai Lake population (Appendix 1) using the DNAsecure Plant Kit (Tiangen Biotech, Beijing, China). A microsatellite-enriched library was developed following the protocol of Glenn and Schable (2005). The genomic DNA was digested into ∼500-bp fragments with RsaI and XmnI (New England Biolabs, Ipswich, Massachusetts, USA) and ligated to the SuperSNX24 adapters (F: 5′-GTTTAAGGCCTAGCTAGCAGAATC-3′, R: 5′-pGATTCTGCTAGCTAGGCCTTAAACAAA-3′). The digestion-ligation mixture was hybridized with 3′ biotinylated oligo probes (AC)18/(AG)18/ (ATG)12 and captured by Dynabeads M-280 streptavidin (Invitrogen, Dynal AS, Oslo, Norway) for enrichment of simple sequence repeat (SSR) sequences. The products were recovered by PCR amplification with the SuperSNX24 forward primer, ligated into the pEASY-T1 Simple Cloning Vector (Transgen, Beijing, China), and then transformed into competent cells of E. coli. Eighty-three positive clones were selected and sequenced with the ABI 3730XL DNA analyzer (Applied Biosystems, Foster City, California, USA). Forty-three clones, or approximately 50% of the positive clones, contained SSRs.
PCR primers were designed for all 43 sequences using the program Primer Premier 5.0 (PREMIER Biosoft International, Palo Alto, California, USA) and evaluated in 20 individuals from six different populations of M. spicatum (Appendix 1). Twenty pairs of primers (Table 1) that showed single and clear bands were chosen and labeled with the fluorescent dyes 6-FAM, HEX, or ROX. Characterization of the SSR loci was estimated in two distant populations in China (Bosten Lake population and Liangzi Lake population; Appendix 1), each with 20 individuals. PCR amplifications were performed in 15 µL total volume containing ∼50 ng genomic DNA, 0.33 µM of each primer, and 1× PCR Mix (Tiangen Biotech). Microsatellites were amplified under the following conditions: 5 min initial denaturation at 94°C; 35 cycles of 30 s at 94°C, 30 s at 52—60°C (Table 1), and 1 min at 72°C; and a final extension at 72°C for 10 min. PCR products were analyzed on the ABI 3730XL and genotyping was performed using GeneMapper version 4.0 software (Applied Biosystems).
Because M. spicatum is hexaploid, up to six alleles per locus should be expressed in one single plant. Of all 20 loci, however, most showed no more than four alleles per individual (Table 2); no reliable explanation could be provided for this considering that the inheritance pattern of M. spicatum was ambiguous. The allele dosage of partial heterozygotes is difficult to identify, thus the presence/absence of the peaks was used to calculate the frequencies for Nei's expected heterozygosity. The locus Myrsp12 showed the highest polymorphism with 13 alleles in the Liangzi Lake population, whereas Myrsp17 and Myrsp20 were monomorphic in both populations. The expected heterozygosity ranged from 0 to 0.873 with a mean of 0.407 and 0.601 in the two populations, respectively (Table 2).
Characteristics of 20 microsatellite markers developed in Myriophyllum spicatum.
Cross-species amplification was conducted in M. verticillatum L. (10 individuals, Appendix 1) and M. sibiricum Kom. (20 individuals, Appendix 1), both of which are in the same section of Myriophyllum as M. spicatum (Moody and Les, 2010). All of the loci were amplified successfully in these two related species.
The polymorphism observed for the microsatellite loci we isolated is high enough to support genetic studies in M. spicatum. Cross-species amplification also reveals that these markers are suitable to use in two related species. We conclude that these primers will facilitate the investigation of genetic diversity, population structure, and evolutionary history of M. spicatum as well as some of its related species.
Results of initial primer screening in two populations of Myriophyllum spicatum.
 The authors thank Song Ge's group for help in molecular techniques. The experiments were partly conducted at the State Key Laboratory of Systematic and Evolutionary Botany, Institute of Botany, Chinese Academy of Sciences. This study was supported by grants from the National Natural Science Foundation of China (30930011 and 31070190).