Genetic diversity in Radix species from the middle and south of Iraq based on simple sequence repeats

Ten newly developed microsatellite loci were isolated from an AG20 and CAG20 enriched genomic DNA library of Radix sp. from Iraq. The simple sequence repeats (SSR) comprised 76.0% di-, 20.0% triand 4.0% tetra-nucleotide repeats. The number of alleles for all 10 loci ranged from 4 to 18, with a mean of 11.3 alleles per locus. The observed (Ho) and expected heterozygosity (He) values varied from 0.286 to 0.927 (mean 0.640) and from 0.263 to 0.939 (mean 0.753), respectively. The polymorphic information content (PIC) values ranged from 0.336 to 0.923 (mean 0.749). Five loci showed significant deviation from Hardy-Weinberg equilibrium (HWE) and no significant linkage disequilibrium (LD) was observed. Cross transferability was successfully tested on species from the genus Melanopsis. These polymorphic SSRs will be useful for assessment of the genetic diversity and population genetics of a Radix sp. as well as other related species in Iraq.

Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License patterns and mating systems (Al-Waaly et al., 2014).Although Glöer and Pešić (2012) considered R. auriculaia to be native to Iraq, and Seddon et al. (2014) mentioned that the specimens from the Southwest Asia are currently assigned to R. auricularia, Naser et al. (2008) found that there was no real presence of this species in the middle and south regions of Iraq.However, there is no information of the genetic diversity of Radix sp. in the middle and south of Iraq.Thus, in order to obtain a better understanding of this species, its genetic diversity needs to be evaluated.
Melanopsis spp. is a freshwater snail, belonging to the family Melanopsidae (Gastropoda: Caenogastropoda: Cerithioidea).This genus is also widely distributed in Iraq (Naser, 2006;Al-Waaly et al., 2014;Mohammad et al., 2014).However, no information on SSR marker and genetic diversity of Melanopsis spp.are available.Although no cross-transferability between families/within order of freshwater snail has been reported, successful transferability of SSR marker has been demonstrated in many studies (Iyengar et al., 2000;Gaur et al., 2003;Dawson et al., 2004).In addition, SSRs have also been widely used in population genetics and phylogenetic studies in insects (Daly-Engel et al., 2012), birds (Parine et al., 2013), fish (Nugraha et al., 2014) and snails (Stoeckle et al., 2014).This study therefore aimed to develop species specific SSR markers for genetic diversity study of rom Radix sp. from the middle and south of Iraq and to apply these newly developed SSRs in Melanopsis spp.

Experimental animals
A total of 30 Radix sp. were collected using a mesh scoop with a wire net of 16 meshes per inch from the middle and south provinces of Iraq, located at Khoura ( 30 36 36 ,47 46 12 ) 1).Radix sp. and Melanopsis spp.samples were preserved in 70% ethanol and genomic DNA was extracted from foot tissue using a Genomic DNA Extraction kit (RBC Bioscience, Taipei, Taiwan) according to the manufacturers' instructions.

SSR-enriched library
Isolation of microsatellite loci from a Radix sp.(AG20 and CAG20) enriched library was performed as described by Sraphet et al. (2011) with some modifications.Genomic DNA of Radix sp.samples was extracted and digested with AluI (Takara, Japan), HaeIII (Takara, Japan) and AfaI (Takara, Japan) restriction enzymes, then purified using the Wizard SV gel and PCR Clean-Up system (Promega, Madison, WI, USA).Purified DNA of each digestion reaction was ligated with linkers and hybridized with biotinylated oligonucleotide probes and subsequently captured using streptavidin-coated magnetic beads (Invitrogen Dynal AS, Norway).Amplified DNA fragments in a size range of 0.5 -1.0 kbp were selected to clone into the pGEM ® -T Easy Vector System (Promega).Recom inant plasmids were transformed into H5αcompetent Escherichia coli cells and plated on LB-agar containing 100 µg/ml ampicillin, 20 mM IPTG and 80 µg/ml of X-gal.Positive colonies containing the expected insert sizes were selected by PCR amplification with vector primers and sequenced.SSR motifs were found using the Web-Sat program (http://wsmartins.net/websat/) (Martins et al., 2009) and redundant sequences were checked using the CAP3 program (http://doua.prabi.fr/software/cap3)(Huang and Madan, 1999).SSR primers were designed using the Web-Sat program.

SSR analysis
Genomic DNA of 30 Radix sp. and three individual samples of each M. costata, M. nodosa and M. buccinoidea were tested with SSR primers.PCR reactions were performed as described by Sraphet et al. (2015) with some modifications, in a total volume of 20 µl containing 15 ng of DNA, 1× Taq buffer, 1.5 mM MgCl2, 0.2 mM dNTPs, 2 µM of each primer and 1 U of Taq DNA polymerase (Thermo Scientific, Foster City, CA).PCR reactions were carried out with denaturation at 94ºC for 2 min, followed by 35 cycles of amplification at 94°C for 30 s, 55°C for 45 s and 72°C for 1 min with a final extension at 72°C for 5 min.The amplified products were analyzed on 5% polyacrylamide gels and visualized by silver staining (Benbouza et al., 2006).

Data analysis
The observed heterozygosity (Ho), expected heterozygosity (He), deviations from Hardy-Weinberg equilibrium (HWE) for each locus and polymorphic information content (PIC) were calculated using genotypic data by PowerMarker V3.25 (Liu and Muse, 2005).The linkage disequilibrium (LD) between all loci was tested using the online version of GENEPOP on the web (Raymond and Rousset, 1995).Cluster analysis and construction of the dendrogram were performed by the Un-weighted Pair-Group Method (UPGMA) using the TFPGA program (Miller, 1997).

Microsatellite characterization
A total of 136 positive clones from a Radix sp.(AG 20 and CAG 20 ) enriched library were selected for DNA

Genetic diversity of microsatellite markers
A total of 50 new SSR primer pairs were designed based on the microsatellite sequences.Of these 50 pairs of SSR primers, 10 pairs showed polymorphisms and these were used to analyze 30 samples of Radix sp., resulting in total of 113 alleles (Table 3 and Figure 2).The number of alleles per locus ranged from 4 (Ra22) to 18 (Ra2), with an average of 11.3 alleles per locus.The observed heterozygosity (Ho) of these polymorphic loci ranged from 0.286 (Ra34) to 0.927 (Ra26) with an average of 0.640, while expected heterozygosity (He) ranged from 0.263 (Ra34) to 0.939 (Ra2) with an average of 0.753.Significant deviation from Hardy-Weinberg equilibrium were observed in this study at the loci Ra2, Ra18, Ra32, Ra42 and Ra49 (P<0.05).Tests for linkage disequilibrium (P<0.001)revealed no linkage disequilibrium among the pair-wise compared loci.In addition, the PIC value of polymorphic SSRs ranged between 0.336 (Ra34) and 0.923 (Ra2), with an average of 0.749.

UPGMA clustering by SSRs
The genetic distance according to Nei (1972) was    5).

DISCUSSION
The SSR-enriched library from Radix sp.showed high efficiency due to the high percentage of positive clones containing insert fragments with the expected size and a reasonable percentage of non-redundancy.While enrichment increased the number of positive clones which contained microsatellite motifs, the level of clone redundancy was still quite high, albeit similar to the redundancy rate found in the study of Zane et al. (2002).
The polymorphism rate of SSR primers developed from Radix sp.enriched library was slightly lower than other species of freshwater snail genus such as Physa acuta (Monsutti and Perrin, 1999), Aplexa marmorata (Dubois et al., 2008) and Bellamya aeruginosa (Gu et al., 2015).
This may be due to the nature of genetic background as well as differences in genetic diversity of this species from each location.In this study, the number of alleles at each locus was higher than previous reports of R. balthica (Salinger and Pfenninger, 2009) and also other freshwater snail libraries such as for L. stagnalis (Knott et al., 2003), Physa marmorata (Dubois et al., 2008), Bulinus forskalii (Gow et al., 2001) and B. aeruginosa (Gu et al., 2015).The PIC (value of polymorphism) detected by a marker (Nagy et al., 2012), in this study revealed high informative levels of the SSRs as described by Botstein et al. (1980).This information and the high mean values of Ho (observed) and He (expected) indicate a high level of genetic diversity of the Radix sp. in Iraq.However, the value of Ho was lower than He suggesting that an inbreeding situation likely occurred resulting in a deficit of heterozygosity within the Radix sp.population.In addition, loci showed significant deviations (P<0.05) from Hardy-Weinberg equilibrium indicating the presence of null alleles at theses loci (Ducarme et al., 2008).No evidence for linkage disequilibrium was detected for these loci suggesting that there is no linkage for all pairs  of loci examined (Salinger and Pfenninger, 2009).
Previous studies have reported that SSR markers are able to cross-amplify loci in related species/genera in plants and animals (BarbarÁ et al., 2007).Successful amplification using SSR between families was about 33% which was lower than comparing between species and between genera (BarbarÁ et al., 2007).No reports of cross amplification across families in freshwater and land snails are available.Radix sp.belongs to family Lymnaeidae, while Melanopsis spp.belongs to family Melanopsidae.Polymorphic SSR primers from Radix sp. in this study were assessed for their transferability to the genus Melanopsis; M. costata (40%), M. nodosa (60%) and M. buccinoidea (40%), and showed to be fully transferrable across families.The results showed that one M. buccinoidea (No. 37) was grouped with Radix sp., while samples No. 9 of Radix sp. was included with Melanopsis spp.This may be due to missing data of some loci that could affect the clustering; however, most of the samples were clustered together in the same genus.This is the first report of cross-amplification between families of freshwater snails and indicates that the newly developed SSRs are able to be applied as an assessment of the genetic diversity in Melanopsis sp.In addition, the markers might be applicable to other groups of freshwater snails.
The SSR markers developed in this study showed a high level of polymorphism in Radix sp.collected from the middle and south of Iraq, as well as with freshwater snails of genus Melanopsis.This study carried out for the first time the genetic diversity and relationship among Radix sp. and high genetic variation within this species in individuals from the middle and south of Iraq was observed.The study demonstrated that SSR markers are useful for further study of the population genetics and evolutionary relationship of Radix sp. and other related species.

Figure 3 .
Figure 3.The UPG A dendrogram ased on ei's genetic distance showing the genetic distance etween Radix sp. and Melanopsis spp.from the middle and south provinces of Iraq.

Figure 4 .
Figure 4.The UPG A dendrogram ased on ei's genetic distance showing the genetic relationship between Radix sp. and Melanopsis species.

Table 1 .
Description of Radix sp. and Melanopsis spp.collection sites from the middle and south of Iraq.

Table 2 .
Type and number of microsatellite motifs from Radix sp.
Figure 2. The example of SSR polymorphic pattern (Ra 42) in Radix sp.

Table 4 .
Specific observed allele in Radix sp. and genus Melanopsis.(* indicates genotype found in specific species tested in crosstransferability of SSRs.).Radix sp. and Melanopsis spp.UPGMA clustering based on Nei's method showed that most of Melanopsis spp. was clustered together and clearly separated from Radix sp.except M. buccinoidea (No. 37) as shown in Figure 3. Radix sp.(No. 9) was included with Melanopsis spp.cluster.Among the group of Radix sp.samples and the Melanopsis genus, UPGMA clustering was divided into 2