Isolation and Characterization of 21 Microsatellite Loci in Cardiocrinum giganteum var. yunnanense (Liliaceae), an Important Economic Plant in China

Twenty-one microsatellite markers from the genome of Cardiocrinum giganteum var. yunnanense, an important economic plant in China, were developed with a fast isolation protocol by amplified fragment length polymorphism of sequences containing repeats (FIASCO). Polymorphism within each locus was assessed in 24 wild individuals from Gaoligong Mountains in western Yunnan Province, China. The number of alleles per locus ranged from 2 to 4 with a mean of 2.9. The expected and observed levels of heterozygosity ranged from 0.042 to 0.726 and from 0.000 to 1.000, with averages of 0.44 and 0.31, respectively. These polymorphic microsatellite markers should prove useful in population genetics studies and assessments of genetic variation to develop conservation and management strategies for this species.

Cardiocrinum giganteum var. yunnanense is a perennial herb and mainly distributed in Gansu, Guangdong, Guangxi, Guizhou, Henan, Hubei, Hunan, Shaanxi, Sichuan, and Yunnan provinces of China and adjacent regions of Myanmar [3]. It grows in forests at altitudes ranging from 1200 to 3600 m elevation [3]. With its attractive flowers (Figure 1), this species has attracted the attention of botanists and horticulturalists, who have taken an interest in the commercial development of this species as an ornamental resource [4]. It is an important economic plant in China. The seeds are used as a replacement for Aristolochia fruits to treat cough [5], and bulbs are used as a starch staple by the local people in Guangxi and Yunnan [6]. Its economic attributes have made Cardiocrinum giganteum var.
yunnanense at risk of overexploitation of natural populations. Moreover, its habitat has been badly degraded and fragmented due to heavy logging and forest destruction in past decades, which has reduced the distribution of this species to a fragmented range with small populations [7]. To provide effective conservation and management strategies for this important economic plant, it is necessary to understand the spatial genetic structure, genetic diversity, and levels of gene flow within and among its populations. However, in the genus Cardiocrinum, except 13 microsatellite loci were developed from the genome of C. cordatum [8], no nuclear microsatellite primers or other types of markers have been reported for C. giganteum var. yunnanense. Microsatellites show numerous advantages over other fingerprinting methods such as RAPD, ALFP, and ISSR because they are locus-specific, codominant, highly reproducible, and usually highly polymorphic [9]. Hence, we have developed and characterized 21 microsatellite markers for Cardiocrinum giganteum var. yunnanense, which will facilitate further investigations on the genetic diversity, population structure, and gene flow of this species.

Results and Discussion
In total, 273 positive clones were sequenced. A total of 223 (82%) sequences were found to contain simple sequence repeats (SSRs); 114 of these with appropriate microstellite and sufficient flanking regions were selected to design locus-specific primers. Polymorphisms of all 114 microsatellite loci were assessed in 24 wild individuals. Of these primers, 30 successfully amplified the target regions, and 21 of them displayed polymorphisms and 9 showed monomorphism ( Table 1). The number of alleles per locus ranged from 2 to 4, with a mean of 2.9. The expected (H E ) and observed (H O ) heterozygosities ranged from 0.042 to 0.726 and from 0.000 to 1.000, with average of 0.44 and 0.31, respectively ( Table 2). Among 21 microstellite markers, 16 loci showed significant deviation from Hardy-Weinberg equilibrium (HWE) (P < 0.01) ( Table 2), probably due to heterozygote deficiency or the limitation of sample size, also because of the presence of null alleles. Twenty-eight loci pairwise (13.3%) showed significant genotypic linkage disequilibrium (LD) between pairs of loci (P < 0.001).

Isolation of Microsatellite Loci
Genomic DNA was extracted from silica-gel-dried leaves by following a CTAB method and the microsatellite loci were isolated by using the fast isolation by AFLP of sequences containing repeats (FIASCO) protocol [10,11]. Approximately 500 ng of total genomic DNA was digested with MseI enzyme (New England Biolabs, Beberly, MA, USA), and then fragments were ligated to the MseI AFLP adaptor pair (5'-TACTCAGGACTCAT-3'/5'-GACGATGAGTCCTGAG-3') at 37 °C for 2 h with T 4 DNA ligase (Fermentas, Burlington, ON, Canada).
The purified PCR products with enriched microsatellite repeats were ligated into the pGEM-T vector (Promega, USA), and transformed into DH5α cells (TaKaRa, Dalian, China). Recombinant clones were screened by blue/white selection and the positive clones were tested by PCR with (AC) 10 /(AG) 10 /(AAG) 7 and T 7 /Sp 6 primers. The clones with positive inserts were sequenced with an ABI PRISM 3730XL DNA sequencer (Applied Biosystems, Foster City, CA, USA). The program Oligo 6.0 was used to design locus-specific primers for those microsatellite sequences found to containt sufficient flanking regions [12].  (Table 1), for 30 s, 72 °C for 60 s, and a final extension step at 72 °C for 7 min. The amplified fragments were separated on 6% polyacrylamide denaturing gels with a 20-bp ladder molecular size standard (Fermentas, Burlington, Ontario, Canada) by silver staining.

Data Analysis
Standard genetic diversity parameters of polymorphic loci, e.g., the number of alleles (N A ), and expected (H E ) and observed levels of heterozygosity. We also estimated deviations from Hardy-Weinberg equilibrium (HWE) and genotypic linkage disequilibrium (LD) between pairs of loci using Chi-square tests.

Conclusions
The 21 microsatellite markers developed in this study are the first set of such markers for Cardiocrinum giganteum var. yunnanense. They should prove useful for further investigating the spatial genetic structure, genetic diversity, and levels of gene flow within and among populations of this species, which will help to develop viable strategies for the conservation and management of this important economic plant.