Genetic differentiation between the black skinned and white skinned snails ( Archachatina marginata ) using random amplified polymorphic DNAs

The study investigates the genetic differentiation between the black skinned and white skinned ectotypes of the giant African land snails (Archachatina marginata) from Cross River State in Niger Delta region of Nigeria. The random amplified polymorphic DNA (RAPD) technique was employed in this study. Five (5) oligonucleotide primers (OPAD-09, OPAE-04, OPAE-05, OPAF-07 and OPAF-09) were used to amplify DNA from five samples of A. marginata constituting black skinned and white skinned ectotypes. A total of 58 RAPD bands with 24 polymorphic bands (40.18%) with size range of 150 to 5,500 bp, were scored from the populations. The black skinned ectotype had mean percentage polymorphism of 34.50%, while the white skinned ectotype recorded mean percentage polymorphism of 41.40%. Genetic similarity coefficient ranged from 60 to 63%, while the genetic distance ranged from 0.37 to 0.40. The genetic similarity between the two ectotypes of A. marginata from Cross River State is high and depicted low genetic differences. This reveals that the genetic variability of the species (A. marginata) from Cross River State is gradually eroding. Efforts should be made to conserve the genetic pool of this species, as the erosion of genetic variability is dangerous and could continue unnoticed till extinction is imminent.

A. marginata offers an opportunity to complement the supply of animal protein from conventional and regular sources such as beef, pork, chevon, mutton, among others.This will to a great extent reduce the problem of animal protein inadequacy, as the per capita protein consumption of an average Nigerian is still low (World Bank, 2001).However, several workers (Ojating, 2001;Ojating and Ogar, 2002;Okon et al., 2008;Okon et al., 2009) have reported a drastic depletion of populations of GALS owing to excessive and indiscriminate gathering of snails (both juvenile and adult) from the wild; coupled with deforestation, bush burning and other human activities.Consequently, the genetic diversity of GALS *Corresponding author.E-mail: blessedbassey4destiny@yahoo.com.Tel: +234 08034183263.(A.marginata) is gradually eroding thus threatened with extinction.Puurtinen et al. (2004) also noted that genetic threats associated with small population size may contribute to the elevated extinction risk of small and isolated populations.This necessitates a swift study to provide genetic informations on GALS (A. marginata) as there is presently little or no information on genetics differentiation and breeding of GALS.
The study of the structure and function of genes at molecular level in a breeding population can help to determine the genetic differentiation and similarity of such population (Lee et al., 2011).One of the modern marker techniques for such study is random amplified polymorphic DNAs (RAPD) (Williams et al., 1998).The technique is simple and does not require prior knowledge of the genome.Also, it requires small amount of DNA (Hadrys et al., 1992).The technique also enables detection of polymorphism in closely related organisms.The objective of this study was to genetically differentiate between the black skinned and the white skinned ectotypes of A. marginata using RAPD.

Collection of samples
A total of 60 snails (A.marginata) comprising of 40 black skinned and 20 white skinned ectotypes were randomly picked from the three agro-ecological zones (Southern agro-ecological zone, Central agro-ecological zone and Northern agro-ecological zone) of Cross River State, Nigeria.

DNA extraction and RAPD
Live snails were transferred to the molecular laboratory of Federal University of Agriculture, Abeokuta (FUNAAB) for genotyping.DNA was extracted following a modified phenol chloroform extraction and ethanol precipitation as described by Sambrook et al. (1989).About 10 µl of each DNA was taken into eppendorf tube with 90 µl sterile distilled water to obtain 20 -50 ng/µl.The total reaction volume of 20 µl was used with final concentration containing 20 ng of genomic DNA, 10x reaction buffer (2.0 µl), 25 mM magnesium chloride (1.6 µl), 5% Tween 20 (2.0 µl) water (8.2 µl).The amplification was programmed at 45 cycles for 20 s of denaturation at 94°C, 20 s of annealing temperature at 37°C, 40 s of primers extension at 72°C and final extension of 7 min at 72°C.This process was repeated four times to ensure accuracy.Five RAPD primers (Table 1) from Operon Kit A (with 60% G + C content) were used.PCR product was electrophoresed on 1.4% agarose gel in 1 x TAE buffer at 150 V and 0.5 miniamp for 2 h.The gel was stained in 2.5 mg/L ethidium bromide for 3 min and further destained for 10 min in sterile water and then viewed under ultra violet light and the picture was taken.

Data analysis
The RAPDs banding profiles were visually scored for all the DNA samples and for each primer.The recording of the data was according to the presence/absence criterion.Clear and visible bands were marked as present (1) or absent (0).Bands that were faintly stained were not considered in the data.These bands were considered as polymorphic when they were absent in some samples in frequency greater than 1% (Jorde, 1995) and change in band intensity was not considered as polymorphism.Similarity coefficients were calculated across all the possible pair wise comparisons of snail samples among populations, using the formula below: 2nxy nx + ny Sxy = Where, nxy is the number of common bands shown in both individuals x and y, and nx and ny are the total numbers of bands observed in individuals x and y respectively (Nei and Li, 1979).As means of providing a visual representation of genetic relationships, a dendrogram was constructed based on the similarity coefficient values (1 -Sxy) between pairs of snail samples.The dendrogram was constructed using the unweighted pair group method of Arithmetic averging (UPGMA) employing the sequential, agglomerative, hierarchical and nested clustering (SAHN) from NTSYS-PC program (Rohlf and Marcus, 1993).

Generation of RAPD pattern of Genomic DNA
Five primers from the Operon Kit A (OPAD-09, OPAE-04, OPAE-05, OPAF-07, OPAF-09) with 60% G+C content were used in this study.These primers were used to generate RAPD pattern of genomic DNA for samples of snails (A.marginata) from Cross River State, Nigeria.The results showed that the largest fragment was 5,500 bp, while the smallest fragment was about 150 bp in size (Table 1).
The results of the RAPD DNA fragment showed that different primers generated different numbers of fragments and lengths of DNA amplification products (Table 2).From the five samples, a total of 58 bands were generated using the five primers.Primers OPAD-09 and OPAE-04 each generated 13 fragments; primer OPAE-05 generated 10 fragments, OPAF-07 generated seven fragments, while OPA-09 generated 15 fragments (Figure 1).
The numbers of RAPDs bands generated by the five primers and in each of the two ectotypes of A. marginata studied are shown in Table 3.The total numberof polymorphic bands were 24 out of 58 reproducible bands     3).This high polymorphism in white skinned ectotype of A. marginata could point towards a moderately higher population of white skinned ectotype and thus higher diversity than black skinned ectotype in the state (Puurtinen et al., 2004).According to Uboh et al. (2010), there is a cultural discrimination against consumption of white skinned snails in some parts of Cross River State, Nigeria.This implies that more of the black skinned ectotype is removed from the wild than the white skinned ectotype, thereby explaining the higher population of white skinned snails in the state.
The similarity coefficient between the white skinned and black skinned snails ranged from 60 to 83% (Table 4) which are quite high.This showed that the genetic differences between the two snail ectotypes is moderately low.This is in agreement with the findings of Wan et al. (2009) who noted that high genetic similarity indicates low genetic differences or variability between snail species.These low genetic differences between the white skinned and black skinned A. marginata could be attributed to a relatively small population size of these snails in the state of study, which might have resulted in high level of inbreeding.
This confirms the findings of Ojating (2001), Ojating and Ogar (2002), Okon et al. (2008) and Okon and Ibom (2012) that the excessive and indiscriminate gathering of GALS from the wild, couple with deforestation, bush burning have led to depletion of GALS population, thus subjecting the genetic diversity of the snail to threat of extinction.The low genetic variability or high genetic similarity could also be as a result of high rate of selffertilization in these snails (Charlesworth et al., 1993;Worth et al., 1997).Furthermore, low genetic variability is reported to arise from reduction in effective population size which then allow further accumulation of deleterious mutations, thus reducing the fitness of the population (Puurtinen et al., 2004).The dendrogram (Figure 2) revealed that the snail samples were not just grouped based on spatial distribution but also based on the genetic closeness.This is indicated in the second cluster where the white skinned sample (5) snail is grouped along the black skinned snails' samples (2) and ( 4).This implies that the black skinned and white skinned A. marginata are of the same genetic background, otherwise there would have been distinct cluster for the white skinned snail.
Thus, the genetic differences here could be due to environmental factors such as random drift, mutation and possibly, selection (Weiner, 1994;Akinnusi, 2004;Puurtinen et al., 2004;Okon et al., 2008;Okon and Ibom, 2012).The genetic distance estimates between black skinned and white skinned A. marginata ranged from 0.37 to 0.40.
This small distance estimate may indicate population substructure like subpopulations in which there is random mating, but between which there is reduced amount of gene flow (Brent, 1996;Wan, 2009).

Conclusion
The genetic differentiation between samples of black skinned and white skinned snails (A.marginata) from Cross River State is low.This is depicted in the high genetic similarity (60 to 83%).Thus, it can be inferred that the genetic differences between these snails is mostly based on the influence of the environment (random drift, mutation and selection), since the snails are of the same genetic background.The results also demonstrated that loss of genetic variability or low genetic differences between these snails is associated with drastic decrease in population size, high level of inbreeding, high level of self-fertilization and thus limited gene flow.The gradual erosion of genetic variability is dangerous, because it may go unnoticed, especially since the population is regulated by ecological factors, until the reproduction rate of the population is so low that extinction is unavoidable.Hence, more research should be carried out towards the conservation of genetic pool of GALS.

Figure 2 .
Figure 2. Neigbouring-joining dendrogram showing the genetic relationship between populations of white skinned and black skinned A. marginata in Cross River State of Nigeria.

Table 1 .
Primers and size of fragments generated.

Table 2 .
Code sequence, nucleotide lengths and G+C content of primers used in the RAPD analysis.

Table 3 .
The number of RAPD fragments and polymorphic fragments generated by five primers in black skinned and white skinned A. marginata.

Table 4 .
Similarity coefficient between black and white skinned snails (A.marginata).