Development of New Microsatellite DNA Markers from Apostichopus japonicus and Their Cross-Species Application in Parastichopus parvimensis and Pathallus mollis

Twenty microsatellite DNA markers were developed for sea cucumber and used to investigate polymorphisms of 60 wild Apostichopus japonicus individuals collected from China. It revealed that all the markers were polymorphic. A total of 164 alleles were detected at 20 loci. The number of alleles per locus varied from 3 to 17 with an average of 8.2, and the expected heterozygosities of each locus ranged from 0.03 to 0.89 with an average of 0.64. Cross-species amplification was also conducted in Parastichopus parvimensis collected from the United States and Pathallus mollis collected from Peru. The result showed that 17 loci amplified Parastichopus parvimensis DNAs while only 4 loci could amplify Pathallus mollis DNAs. All of the polymorphic markers would be useful for future genetic breeding and the assessment of genetic variation within sea cucumbers.


Introduction
Sea cucumber (Apostichopus japonicus) belongs to the phylum Echinodermata, class Holothuroidea. It is a common species distributed in shallow waters along northern Asian coasts in China, Japan, Russia and Korea [1,2]. It is the most valuable species in China due to its high nutritional and medicinal properties. However, resources of natural sea cucumber in Asian coast have decreased dramatically due to overfishing and severe environmental pollutions. Artificial propagation techniques for sea cucumber were developed in China in the 1980s. In recent years, sea cucumber aquaculture has developed rapidly, and the production reached 102,159 tons valued at 20 billion Yuan in 2009 [3]. However, due to inappropriate broodstock management, low-grade artificial seeds have caused tremendous decrease of genetic diversity and deterioration of disease resistance and growth performance. Thus, it is very important to carry out research on sea cucumber genetics, such as establishing broodstock for selection, estimating genetic parameters for growth and anti-bacteria, establishing genetic linkage map and development of molecular markers for species identification and population genetics studies.
To date, genetic marker systems including allozyme [4,5], AFLP [6], microsatellite [7][8][9][10], ISSR [11], mtDNA [12] and SNP [13] have been used in genetic studies. Of all the molecular marker types, microsatellite markers have been proven to be an extremely valuable tool for genetic studies and conservation and management of genetic resources. However, only 65 microsatellite markers developed from an enriched microsatellite library have been reported in A. japonicus until now [7,14]. Additional highly informative microsatellite markers are necessary to investigate the status of the wild resources and the genetic linkage map construction.
In the present study, we reported development of microsatellite in sea cucumber (A. japonicus) and characterization of the microsatellite markers by genotyping 60 individuals sampled from a wild population. Additionally, cross-species amplification was carried out to determine the potential for cross utility, and amplification of identified markers were assessed in two related species-warty sea cucumber (Parastichopus parvimensis) and black sea cucumber (Pathallus mollis).

Results and Discussion
Out of 252 random selected recombinant clones, 142 clones (56.3%) were found to contain inserts with a microsatellite motif in the middle position indicating that the enrichment was highly effective. Of the 142 clones surviving PCR screening, 118 (83.1%) contained a microsatellite motif in the middle position after being sequenced (Table 1). According to Weber's (1990) classification rules [15], the sequences were divided into three categories: 83 perfect repeat sequences without interruptions in the runs of CA or GT di-nucleotides (70.4% of total), 30 imperfect repeat sequences with one or more interruptions in the run of repeats (25.4%), and 5 compound repeat sequences with adjacent tandem simple repeats of a different sequence (4.2%). When classified using the repeat sequence type, 116 clones (98.3%) had di-nucleotide repeats among which 88 clones' repeat number was between 5 to 9 and 28 clones' repeat number was higher than 10, the other two clones had tetra-nucleotide motifs. As the first batch, twenty pairs of primers were designed according to 30 sequences and used to investigate polymorphism of 60 sea cucumber individuals. All of the tested 20 primer pairs showed clear band patterns and polymorphic ( Table 2). A total of 164 alleles were detected at 20 loci and the total of effective allele numbers was 84. The number of alleles (N a ) at each locus ranged from 3 (AJ08) to 17 (AJ07) with an average of 8.2. The effective allele number (N e ) ranged from 1(AJ08) to 8.3 (AJ01) with an average of 4.2. The difference between Na and Ne was caused by the uneven frequency of each allele. As to the relationship between the number of microsatellite repeats and polymorphism, many scholars have different views. Qu suggested that the polymorphism would be higher when the number of microsatellite repeats increased [16]. Zheng considered that many high polymorphic loci would be missed if only loci with high number of microsatellite repeats were chosen in genetic research [17]. The result of this experiment showed that there is no relationship between the number of microsatellite repeats and polymorphism, which was consistent with Zheng.
The observed heterozygosities (H o ) of each locus ranged from 0.02 (AJ10) to 0.79 (AJ09) with an average of 0.38, and the expected heterozygosities (H e ) of each locus ranged from 0.03 (AJ08) to 0.89 (AJ01) with an average of 0.64. According to the polymorphic index content (PIC) value of each locus, three loci (AJ04, AJ08 and AJ09) were low polymorphic (PIC < 0.25), three loci were moderate polymorphic (0.25<PIC < 0.5), and the other 14 loci were high polymorphic (PIC > 0.5). None of the loci showed significant linkage disequilibrium. After sequential Bonferroni correcting for multiple tests, fourteen loci were found to depart significantly from Hardy-Weinberg equilibrium (HWE). Further tests indicated that heterozygote deficiency at these loci was responsible for the departure. Another possible explanation for the departure from Hardy-Weinberg equilibrium is the dramatic decline in spawning populations, and non-random mating or genetic bottlenecks.
Cross-species amplification test showed that only 4 loci were successfully cross-amplified in P. mollis (Table 2), while 17 loci (85%) could get cross-amplification in P. parvimensis. It confirmed that microsatellite markers developed in A. japonicus could be used for related sea cucumber species.

DNA Extraction
Sixty individuals of sea cucumber (A. japonicus) were collected from Qingdao coast in China. Warty sea cucumber (Parastichopus parvimensis) collected from USA and black sea cucumber (Pathallus mollis) collected from Peru in dried hard form were bought from the supermarket. The dried sea cucumbers were immersed in pure water in order to get water raise sea cucumber. Genomic DNA was extracted from longitudinal muscle using Mollusc DNA kit (Omega, USA). The extracted genomic DNA was stored at −20 °C until genotyping.

Isolation of Microsatellite-Containing DNA Fragments and Primer Design
In order to check whether the microsatellite motif located in the middle of the insert, each recombinant was subjected to three individual PCR screening using two universal sequencing primer and (CA) 12 DN oligonucleotide. In the first reaction, universal forward and universal reverse sequencing primer were used; in the second reaction, universal forward sequencing primer and (CA) 12 DN oligonucleotide were used; in the third reaction, universal reverse sequencing primer and (CA) 12 DN oligonucleotide were used. Recombinant clones that produced products in obviously different length between the first reaction and the second or the third reaction were sequenced and trimmed. The sequencing data were scanned using the software SSRHunter V1.3 [19]. Sequences with microsatellite motifs and flanking regions were selected for PCR primer design with the help of Primer Premier [20].

PCR Amplification and Genotyping
Microsatellite primers designed were used to amplify genomic DNA of 60 sea cucumber individuals, 5 warty sea cucumber individuals and 5 black sea cucumber individuals. The PCR mixture contained 1× buffer, 1.5 mmol/L MgCl 2 , 200 µmol/L dNTP (each), 200 µmol/L primers (each direction) and about 50 ng genomic DNA. The PCR conditions were denaturing at 94 °C for 1 min, followed by 30 cycles of 1 min at 94 °C, 1 min at annealing temperature, and 1 min at 72 °C, with a final extension for 5 min at 72 °C. The optimized annealing temperatures of different primer pairs were listed in Tm column of Table 2. The PCR product was separated on a 6% denaturing polyacrylamide gel and visualized by silver staining. Allele size was determined with software Quantity One V4.62 (Bio-rad) by referring to 20bp DNA ladder marker (TaKaRa, Dalian, China).

Genetic Data Analysis
Popgene version 1.32 [21] was used to calculate the number of alleles (Na), the number of effective alleles (Ne), observed heterozygosity (Ho) and expected heterozygosity (He). Polymorphism information content (PIC) of each locus was calculated according to Botsein (1980) [22]. Hardy-Weinberg equilibrium and linkage disequilibrium test were conducted using Genepoponline version [23]. Significance criteria of all multiple tests were corrected following sequential Bonferroni correcting [24].

Conclusions
In the present study, microsatellite-enriched genomic library of sea cucumber (Apostichopus japonicus) was constructed and a total of 20 novel genomic microsatellite DNA markers were developed. Most of the markers could amplify successfully in warty sea cucumber and black sea cucumber. The use of these microsatellite markers will certainly facilitate the management and exploration of the genetic resources of Holothuroidea and assist in their genetic improvement to some extent.