Spartina alterniflora Loisel. (Chloridoideae, Poaceae) is a perennial grass native to the Atlantic and Gulf coasts of North America, and has been used in coastal restoration programs in many countries (Daehler and Strong, 1996). However, S. alterniflora is highly invasive in many parts of the world where it is introduced. In China, the species has been listed as one of the nine most notoriously invasive plants (Zhi et al., 2007). Recently, we have sequenced the transcriptome of S. alterniflora using the next-generation sequencing platform Illumina Genome Analyzer II to understand its invasion in China (Guo et al., unpublished data). The transcriptome sequences contain abundant simple sequence repeat (SSR) markers, which should be very useful in population genetic studies. Here, for the first time, we identified several thousand expressed sequence tag (EST)-derived simple sequence repeat (ESSR) markers from the RNA-seq data of S. alterniflora. Compared to genomic SSR markers, ESSRs are easier and less expensive to develop, as well as more transferable across taxonomic boundaries (Ellis and Burke, 2007).
METHODS AND RESULTS
Transcriptome sequencing of S. alterniflora was conducted using the Illumina Genome Analyzer II system. In total, 14.55 million 90-nucleotide pairedend reads were obtained and assembled into 69 899 contigs with an average length of 503 nucleotides by using two short-read assemblers—Trinity and CAP3 (Huang and Madan, 1999; Grabherr et al., 2011). These unique sequences (i.e., ESTs) were further screened for the presence of microsatellites using MISA ( http://pgrc.ipk-gatersleben.de/misa). A total of 3052 potential ESSRs were identified. The micro satellites were defined as di-, tri-, tetra-, penta-, and hexanucleotide SSRs with a minimum of four contiguous repeat units. The most abundant repeat type was trinucleotide (47.8%, 1460), followed by dinucleotide (38.7%, 1182), tetranucleotide (8.1%, 247), pentanucleotide (3.7%, 114), and hexanucleotide (1.6%, 49) repeat units. Primer3 software (Rozen and Skaletsky, 2000) was used to design 50 primer pairs with an expected product size ranging from 100 to 300 bp. Sixty individuals of S. alterniflora representing five populations in China (Appendix 1) were used to evaluate the polymorphisms of the microsatellite loci.
Genomic DNA from each individual was extracted from silica gel-dried leaves using the cetyltrimethylammonium bromide (CTAB) method (Doyle, 1991). PCR amplifications were performed in a final volume of 20 µL, containing 2 µL 10× PCR buffer, 2 µL of 2 mM each dNTPs, 1.2 µL 25 mM MgCl2, 1 µL 10 pM forward primer, 1 µL 10 pM reverse primer, 2 U Taq DNA polymerase (Sangon, Shanghai, China), and 10 ng of genomic DNA. The PCR reactions were conducted with the following conditions in a thermocycler (Bio-Rad Laboratories, Hercules, California, USA): initial denaturation at 94°C for 5 min, followed by 34 cycles of 94°C for 30 s, 60°C for 30 s, and 72°C for 45 s, with a final extension cycle at 72°C for 10 min. PCR products were electrophoresed on 8% Polyacrylamide denaturing gel and visualized by silver staining. The band size was estimated by comparison with a 20-bp DNA ladder (Fermentas, Vilnius, Lithuania). Twenty-six (52%) of the primer pairs failed to amplify products, two (4.0%) generated complex band patterns that were difficult to genotype, seven (14%) were monomorphic, and 15 (30%) displayed clear polymorphisms (Table 1). To determine the function of polymorphic SSR-associated unigenes, ESSRs were evaluated for connections with genes of known functions; those 22 sequences (including seven monomorphic and 15 polymorphic loci, respectively) were blasted against the GenBank nonredundant database using BLASTX (Altschul et al., 1997) with an E-value of 10-10.
Characteristics of 15 polymorphic and seven monomorphic ESSRs developed in Spartina alterniflora.
All of the sequences showed significant similarities to known genes (Table 1). The allele number (A) and polymorphism information content (PIC) were calculated for each of the loci using a Web-based calculator ( http://www.genomics.liv.ac.uk/animal/pic.html). The number of alleles per polymorphic locus ranged from two to eight, with an average of 3.80 alleles per polymorphic locus. The number of alleles per individual ranged from one to six, which was consistent with the hexaploidy of S. alterniflora. Seven (46.67%) of 15 polymorphic loci showed the disomic pattern with a maximum of two alleles per individual. These 15 polymorphic microsatellite loci are further characterized in Tables 2 and 3. The expected heterozygosity (He) and observed heterozygosity (Ho) for each disomic locus were calculated using POPGENE (version 1.32; Yeh and Boyle, 1997). The Shannon index for each nondisomic locus was calculated using POLYSAT (version 1.2–1; Clark and Jasieniuk, 2011). The He for those loci was also calculated using the Web-based calculator ( http://www.genomics.liv.ac.uk/animal/pic.html), assuming Hardy—Weinberg equilibrium (HWE). In general, S. alterniflora showed a moderate level of genetic polymorphisms in China. The average Horanged from 0.21 to 0.51, and the He ranged from 0.27 to 0.46 based on seven disomic loci. The average Shannon index (I) ranged from 0.26 to 0.94 in eight nondisomic loci. Five disomic loci (including SaESP06 in all five populations; SaESP09 in the Fujian, Shanghai, and Jiangsu populations; SaESP13 in the Taiwan and Hong Kong populations; SaESP18 in the Taiwan population; and SaESP19 in the Fujian population) deviated significantly from HWE (P < 0.05). It is likely that the observed departures from HWE are due to the presence of null alleles or a result of mixed reproductive modes (selfing and outcrossing) in S. alterniflora. The monomorphism at some loci observed for some populations may also reflect nonequilibrium population dynamics resulting from its recent introduction and spread in China (Tables 2 and 3).
In this study, we report the development and characterization of a set of ESSRs, which was derived from a large-scale transcriptome sequencing of S. alterniflora using the Illumina Genome Analyzer II system. Fifteen polymorphic and seven monomorphic microsatellite markers were identified. Seven of those polymorphic loci display disomic inheritance. These newly developed ESSRs should be valuable for population genetic studies of this invasive species, and they can be used as new tools to trace its invasion history in China.
Characterization of seven disomic polymorphic microsatellite loci in Spartina alterniflora.
Characterization of eight nondisomic polymorphic microsatellite loci in Spartina alterniflora.
 The authors thank Lu Xia, Lu Lu, and Xiaoting Fang for sample collections. This study is supported by grants from the National Natural Science Foundation of China (41130208, 91231106, 41276107, 31070290, 31170213), the Science Foundation of the State Key Laboratory of Biocontrol (SKLBC2011A06), the Fundamental Research Funds for the Central Universities (091gpy35, 101gpy20), and Chang Hungta Science Foundation of Sun Yat-Sen University.