Evaluation of Genetic Diversity of Golden Apple Snail, Pomacea Canaliculata

ABSTRACT Genetics is a trend these age especi eachy that, deoxyribonucleic point barcoding has been developed. deoxyribonucleic acid barcoding is an master(prenominal) tool in categorizing the taxa of antithetic species and it tells so much active the species traits, including hereditary smorgasbord. The Pomacea shadoweraliculata was introduced in miscellaneous part of Asia and had been an invasive species and a pest in assorted ecosystems ever since the fundament. In concord this species of snails, samples were still, deoxyribonucleic acids were extracted, on a lower floorg sensation PCR and cataphoresis, and was orderd and analyzed.The analysis was soft In the Philippines bit quantitative in China. In the Philippines, the cytochrome oxidase fractional monetary unit 1 (COI) brokers was apply and comp atomic number 18d among the species collected and when it was sequenced, it showed releases ascribable to localized break, match and non-corres consortium ence of bases. And in China, the smorgasbord was analyzed by means of Neis gene revolution, Shannons discipline index, percentage of polymorphic bands (PPB) an AMOVA anlysis. 2 the quantitative and qualitative showed that at that place was diversification indoors and among the tribes of these snails.INTRODUCTION Genetics is the study of the genes, and the heri circuit board endowment and var. of individuals. Understanding the genome, which is the complete throttle of chromosomes or the entire ge nonype of an individual, is important beca ending it economic aids in the taxonomic categoryomy of species, especi entirelyy straighta eld wherein advancements in science needs more than detail information, and that basing on morphological features is non enough. sustenance is specified by genomes which contain all the biological information which is encoded in its deoxyribonucleic acid (desoxyribonucleic acid) and divided into units or the genes.The genes argon the dra ught for invigoration beca determination it is the particulate determiner of familial traits. Hence, desoxyribonucleic acid barcoding became a trend for scientists and researchers for the dread of the assorted forms in the traits of antithetic organisms. The well-situated apple snails (Pomacea push asidealiculata) originated from the S come forwardh America, Central America, the air jacket Indies and the Southern USA (Pain 1972) and was spread in the past decades to the different move of southeastern Asia, namely Philippines, China, Thailand, Cambodia, Hong Kong, In makesia and Japan.The introduction of the P. canaliculata without prior studies ca employ alter to the different plants and it became an invasive species which emergenceed to becoming pests to valet and competitors to some new(prenominal) local snails, example of which are those from the genus Pili. The P. canaliculata was observed to direct different proceeds and reproduction in different separate of A sia, to giveher with their external characteristics due to the different habitats and environmental conditions (Keawjam, 1986 and 1987), therefore there is the hypothesis to misidentify dickens sympatric species as one.On the other hand allopatric peoples inhabiting different habitats may show ecomorphological divisions and questionable species posture and it was in addition redeed that the golden apple snails had lavishly adaptability accordly it was easier for them to form vernal tribes ( dong et al. 2011). The brain of the genomes of different species is a trend for scientists these age nevertheless(prenominal) the information or so the different mollusks is still limited. The basic information on the number of species and/or people is of help for conservation programs (Carvalho and Ha officer, 1994) and for building appropriate wariness schemes.In contribution, the studies aims are to evaluate the ancestral change of the golden apple snail population in Asia , namely, Philippines and China via molecular(a)ly characterizing the P. canaliculata and to find different ways of analyzing the gathered data from the sequenced desoxyribonucleic acid of the said species. REVIEW OF RELATED writings Genetic diverseness (Reed 2005) The significance of ancestral diversity arose from devil necessities patrimonial diversity is required for populations to evolve in chemical reaction to environmental changes and heterozygosity levels are linked at one time to reduce population fittingness via inbreeding low gear.The center of transmitted variation a population contains is predicted to correlate with current physical physical fittingness and, in the case of heritabilities (which can remain noble school or even increase despite severe reductions in population size) with evolutionary potential. This correlation in the midst of fitness and levels of contractable variation, however, may be weak or nonexistent due to the neutrality of mole cular scars enjoymentd in estimating heterozygosity, nonadditive hereditary variation and the purging of noisome alleles beca hold of change magnitude pickax against homozygotes.There is a body of literature that points that allozyme heterozygosity is a good measure of population fitness and adaptive potential. Others caution though that much(prenominal)(prenominal) molecular genetic data intimatelyly reflect however a minuscule portion of genome and thus may non be indicator of adaptive genetic differences. But molecular markers may be reclaimable for assessing the extent of genetic divagate. Moreover, ruinous alleles, in vicissitude-selection balance, are responsible for at least half of the genetic variation in fitness.Selection has the tendency to purge the population of the deleterious recessive alleles which in surmise creates inbred populations with a high fitness than their outbreed progenitor. In other words, inbred populations with less(prenominal) genet ic diversity would arouse higher fitness if the population is not kept small enough for a liberal enough to allow the fixation of deleterious alleles to occur. physical fitness and future adaptability are cut in smaller populations of plants and animals due to drift and inbreeding depression.Commonly used surrogates for fitness such as heritabilities, heterozygosity, and population size, noteworthyly correlate with fitness and explain 15-20 % of the variation in fitness. Correlations suggest that m whatsoever populations fork over reduced fitness as a result of inbreeding depression and genetic drift. There is much spat and concern thus, over genetic variation because of the fact that endangered species typically assume lower levels of heterozygosity and the loss of adaptive genetic variation and inbreeding depression puts wildlife populations at an change magnitude risk of extinction.Finally, this increase occurs as a result of the reduction of productive fitness because of inbreeding depression or due to the stroke of tracking the change in abiotic and biotic environment of the population as a result of the loss of genetic variation through drift. deoxyribonucleic acid barcoding (Moritz & Cicero 2004) At the real core, the use of goods and services of desoxyribonucleic acid barcoding is for large graduated table screening of one or a few reference genes in iniquityclub to seize unknown individuals to species and enhance uncovering of naked species.In the hope of developing a comprehensive database of sequences that will serve as a comparison tool to sequences from sampled individuals, proponents used desoxyribonucleic acid barcoding. There is, however, nothing hot with desoxyribonucleic acid barcoding as it is an offshoot of the use molecular markers for the very same purpose except, in desoxyribonucleic acid barcoding, there is an increased carapace and proposed standardization. The selection of one or more reference genes characteri zes standardization, with regards to microbial lodge and in stimulating large scale phyletic analyses if of proven value, though whether or not one gene fits all remain to be a question.Presently, most methods of deoxyribonucleic acid barcoding are tree- ground and can make pass into two broadly delineate classes. one and only(a) class is the distance-based, wherein it is based on the dot of DNA sequence variation at heart and between species. This human body of come near converts DNA sequences into genetic distances and indeed uses these distances to establish appellative schemes. It further defines a likeness threshold below which a DNA barcode is appoint to a known or a sweet species. There is also the address by several authors of a barcoding gap, a distance-gap between intra- and interspecific sequences, for species appointment.However, the distance-based approach seems to be ill suited as a general means for species identification and the discovery of new s pecies. One dry land is that substitution pass judgment of mitochondrion DNA vary between and within species and between different sorts of species. The vary substitution rates can result in broad overlaps of intra- and interspecific distances, and hinder the accurate appointee of query sequences. Another class which is the monophyly-based requires the convalescence of species as discrete clades (monophyly) on a phylogenetic tree and is used to assign unknown taxa to a known or new species.Similarly, some issues complicate the use of monophyly in a barcoding framework. For example, the long-recognized problem of unelaborated lineage sorting will leave gene genealogies that may differ in topology from locus to locus. The lately diverging taxa may not be in return monophyletic due to lack of time call for to coalesce. In addition, the gene trees are not necessarily congruent with species trees, and the monophyly, while a discrete criterion is arbitrary with lever to syst ematic level.Moreover, there is a recently applied new technique that has been proposed as an secondary to tree-based approaches for DNA barcoding, the so called character-based DNA barcode method, which is based on the fundamental conceit that members of a given taxonomic group share attributes that are absent from comparable to(predicate) groups. It is the kind of method that characterizes species through a unique crew of diagnostic characters or else than genetic distances. The four standard homes (A,T,C,G) if sight in fixed states in one species can be used as nosology for identifying that species.This way, species boundaries can be defined by a diagnostic set of characters which can be increased to any level of resolution by applying three-fold genes. Presently, character-based DNA barcode method has been proved useful for species identification and discovery of several taxa. In the view that angiotensin-converting enzyme-gene sequence should be the primary quill id entifier of species, a contention arises that if that is the case then therell be a trustworthy need to connect different life history stages and increase the precision and dexterity of field studies involving versatile and difficult-to-identify taxa.Although the DNA barcoding federation has put emphasis on the grandness of big sequence database within the quick framework and practice of systematics, it should be aegir in mind that DNA barcoding is not the primary answer in solvent the tree of life. Furthermore, as much as the term DNA barcoding appealing, it implies, however, that each species has a fixed and invariant characteristic. But this kind of implication renders un peacefulness to the minds of evolutionary biologists.In evaluating thus, the declare and pitfall of DNA barcoding, two areas of screening should be distinguished the molecular diagnostics of individuals relative to described taxa and DNA-led discovery of new species. And although there is little doubt t hat large-scale and standardized sequencing, when integrated with existing taxonomic practice, can contribute importantly to the challenges of identifying individuals and change magnitude the rate of discovering biological diversity as presented by this study, the general utility of DNA barcoding still requires further scrutiny.PCR (Moore 2005) In apace copying a selected template sequence from a DNA mixture in vitro, PCR offers a wide range of applications such as sequence mentionion and closing off for research, forensics and species identification through the PCR itself and in combination with other techniques. PCRs new technique uses flourescent probes to manage the amounted product at end of all cycle and PCR machines look for the cycle at which the can readily detect flourescence.PCR is also being used to monitor ribonucleic acid through the addition of reverse transcriptase enzyme at the beginning to generate DNA template. In addition, there are now new applications of PCR like single nucleotide polymorphism detection and screening. Cytochrome Oxydase subunit 1 (COI) (Buhay 2009) COI plays a significant role in documenting biodiversity and remains to be the choice for phylogenetic and phylogeographic studies. COI is a mitochondrial protein-coding gene which is a wide accepted marker for molecular identification across diverse taxa.Mitochondrial DNA (mtDNA) go a relatively fast mutation rate, thus they result in significant differences between species. With respect to this, the mitochondrial cytochrome c oxidase subunit I (COI) gene with 700bp was proposed to be a potential barcode or marker for molecular identification across various taxa. Furthermore, COI is a protein coding gene that has an open drill frame and in thecase of barcoding, COI can be exceedingly divergent from the actual COI sequences which may cause major problems because species identification is based on sequence similarity.Pomacea canaliculata (Cowie 2002) The Pomacea canali culata belongs to the family Ampullariidae. Its structure appears to have a slight dimorphism in var. of aperture and operculum. Females have broader lip and concave operculum while convex in male. In footing of reproduction, oviposition often takes place at night or at early aurora or evening about 24 hrs afterward copulation up to two weeks after mating (occurs three generation per week) which occurs anytime of the day or night although there may be some diurnal rhythm.On each oviposition occasion a single clutch is laid of highly multivariate egg number. Moreover, the interval among successive ovipositions for p. canaliculata has been inform to be about five days and hatching generally takes place about two weeks after oviposition. The P. canaliculata breeds only during pass and grows into maturity in less than two months. P. canaliculata is said to be prolific and hence has rapid succession of generations which leads to rapid population expansion.They relatively inhabit still urine and in pissing temperatures above 32 head Celsius, it has been observed that the mortality of p. canaliculata is high. Whereas in low temperature p. canaliculata can survive 15-20 days at 0 degree Celsius, 2 days at -3 degree Celsius but only 6 hrs at -6 degree Celsius. And it is sufficiently tolerant of sea water to survive long enough to be carried by currents from one stream mouth to another, thereby expanding its distribution. P. canaliculata shows preferences among food plants.Its rate of harvest has a direct correlation with its eating on the preferred plant. Moreover, it is able to detect its food plants from some distance utilize chemical cues in the water. P. canaliculata, however, appears to be relatively generalist and indiscriminate that it is viewed to be oddly voracious compared to other Ampullariids. METHODOLOGY try Snail samples were set and collected from 2 countries in Asia, specifically in the Philippines and in China, where the P. canalicula ta was introduced. In the Philippines Los Banos (Dong et al. 011, p. 1778), 2 barangays in Tarlac (Brgy. Cabayaoasan, pan outiqui and Brgy. Pance, Ramos) and Iloilo (Chichoco & Patdu 2012, p13), 44 snail samples were collected. And in China, specifically from Yuyao and Taizhou in Zhejiang province, Fuzhou in Fujian province, Guangzhou in Guangdong province, Nanning in Guangxi province, Kunming in Yunnan province, wherein a total of 120 samples were identified with the conserved sequence by Matsukura et al. (2008) and Pan et al. (2009) and then was collected (Dong et al. 2011, p. 1778).The snails were then stored, any by wrapping in paper, freeze or preserving it in ethanol, and brought into the individual labs in each country for the next move DNA blood, PCR, electrophoresis and sequencing. DNA extraction The two studies used the phenol-chloroform method (Bergallo et al. 2006) with an alternative of the Qiagens Dneasy extraction kit for China. The DNA concentration was determi ned spectrophotometrically and adjusted by a mini- gelatin method (Maniatis et al. , 1982) when the extracted DNA was enough, it was stored at 4oC to -20oC until needed. PCR and ElectrophoresisThe PCR method was basically done by choosing the right primers that will yield understandably reproduced bands and they time-tested the proper amounts and enlargement do of the components of PCR, which were the Mg2+, dNTPs, DNA templates and polymerase, and the primers. After the mixture of the components and the DNA extracted, it was carried out in the thermocycler programmed for pre-denaturing at 94C for 3 min, followed by 26 cycles of 94C for 10-30s, 36-52C for 30-45s, continuation of 65-72C for 60-90s, and the final university extension for 5-7mins at 72C for final extension with 38-48 cycles.After which, the amplified products together with negative controls were run in electrophoresis to be separated and tested for contaminations, respectively. The products were then purified subs equently on with the respective kits present in each lab. In the Philippines, the reaction was done with 2? L MgCl2, 5? L PCR buffer, 1? L dNTP, 2. 5 ? L of the primers, which were the LCO1490 and HCO2198, distilled H2O with 22. 75 ? L, 0. 25 Taq, and 10 ? L Q-buffer. The electrophoresis was done after the ethidium bromide espial (Maniatis et al. , 1982), analyzed through 1. % agarose gels and visualized under a transilluminator. In China, they made use of the ISSR-PCR analysis where they got four primers, which produced clearly reproduced bands, out of the 90 that was screened from the University of British Columbias primer set and the reactions were done with a volume of 20 ? l, containing 0. 2 mM of each dNTP, 1. 5 mM MgCl2, 0. 5 ? M primers, 1 U Taq polymerase and 10 ng DNA template, and also with the determination of the optimal reaction system of ISSR for P. canaliculata (Dong et al. 2011, p. 1779).The products sizes after the amplification was estimated using DNA marker DL20 00 and then was run in electrophoresis, which was done on 6% polyacrylamide gels, visualized by silver staining and then photographed (Li et al. , 2009). Sequencing/ data analysis Chichioco and Patdu (2012) sent the DNA samples to the inaugural Base science laboratory in capital of Singapore for sequencing and the results were sent back to the DNA barcoding science laboratory after a week. The COI sequences were align in the BLAST, specifically the STADEN package variation 1. 5. 3 and Bioedit Sequence Alignment Editor version 7. 0. 9. 0.Aside from the sequences sampled, other sequences and their haplotypes from the GenBank were also compared and matched. In Dongs (2011) research, he made use of the RAPD fragments by labeling them into binary matrices, used them to get the similarity index, Sxy = 2nxy / nx+ ny, where nx and ny represent the number of RAPD bands in individuals x and y, and nxy represents the number of shared bands between individuals, as stated by Nei and Li (197 9), then averaging it across all the realistic comparisons between individuals within a geographic sample to get the within samples similarity (Si).Between sample similarity corrected by within sample similarity Si and Sj of geographic samples i and j, respectively) is also compute between pairs of individuals across samples i and j using the equation Sij = 1 + Sij (Si and Sj)/2. Genetic distance between opposite samples was then calculated as Dij = 1- Sij (Lynch, 1990). RESULTS AND DISCUSSION In the Philippines (Chichioco & Patdu 2012, p. 18-31) The collected samples from Brgy. Cabayaoasan were found in the elevated parts of a rice paddy, specifically, it was a dreary substrate with decaying leaves from the rice plants and surrounding trees while those that was found in Brgy.Pance was in the dim bottom of the shallow fish pond in the roots of water lilies and grasses. The samples from the two barangays in Tarlac and Iloilo had relatively different color in and sizes. Those t hat were collected from Brgy. Cabayaoasan had the largest size and they are slanting black while those in Brgy. Pance had chocolate-brown in color and still, those in Iloilo had very small sized specimens and some of the specimens can be mistaken as Pila conica snails if not examined properly. Primers affect the amplification success greatly, since according to Hajibabaei (2005) a 95% success is requisite for barcoding.The primers LCO1490 (SENSE) and HCO2198 are generally used for the amplification of forward and reverse fragments from COI genes. The DNA samples were subjected to the PCR and agarose gel electrophoresis (AGE), and they produced single discrete bands that suggest that the fragments were homogenous and start and end at the same point (Reece 2004). The bands that were brighter and distinct are more appropriate for sequencing because it means that the DNA fragments were well amplified. The best DNAs were elect and forwarded to the First Base Laboratory in Singapore fo r sequencing.At the return of the results, other sequenced DNA barcodes were also collected and was aligned and compared with the Basic Local Alignment count Tool (BLAST) database. figure of speech 1. Alignment of the COI gene sequences of the Pomacea canaliculata (CPT1-5 from Brgy. Cabayaoasan, PRT 7,9,10 from Brgy. Pance, IICK & IIPC1,3 from Iloilo) from the Philippines using Bioedit Sequence Alignment and ClustalW sextuple Alignment (Chichioco & Patdu 2012, p. 26) By aligning the sequenced data, it can be seen that there are two similarities and differences among the genetic make-up of the samples.The differences are due to localized gap, mismatch and non-correspondence of bases along the COI fragments as pointed out in fig. 2 Fig 2. Comparisons of the COI sequences of the P. canaliculata samples from 35bp- 120bp (Chichioco & Patdu 2012, p. 28) As emphasized in fig. 2, on the 55bp-58bp, a sequence from CPT1 was observed having (5-AATT-3) while all the others have (5-GGTA-3). Even though this is a noticeable difference and could have been caused by mutation or variation, the difference is still low enough and less that 1% difference to be considered significant.But on the other hand, the PCa1 sample had 36 different base pairs compared with the others, which was 5. 5% difference and is high enough and can be considered significant since it is 4% divergent(Meyer and capital of Minnesotaay 2005). Then with a 98% confidence, it could be said that PCa1 is from an independent evolutionary lineage and might indicate a divergence within or extraneous its population or might have occurred due to relationships and interactions among the other species.The introduction of the P. canaliculata to different places may have an effect on its intra- and interpopulation and might be why it has various genetic sequences although it goes against the theory that introduced species becomes a launch population in a new habitat thus they have a limited gene pool and as a cons equence genetic drift, which removes discrepancy since it affects all genes, and bottleneck might occur, which reduces the new species to have a reduced genetic diversity.To observe the genetic variability, the sequences collected were compared with those from GenBank with the use of the BLAST software. As a result from 81 COI barcodes and 55 haplotypes, the samples collected showed 99% and 100% similarities with the different haplotypes thus it showed that the species has a high diversity within the populations. The phylogeography within and among the species does not apply on the P. analiculata since intra- and interpopulation diversity was observed which was shown by the multiple introduction throughout the Philippines, hence the different time of the introduction contributed more to its diversity and it coincides with the unsettled pool model that says that the introduced population acquires more genetic variability because of the multiple sources of genetically divergent popu lations as compared to that of the local species (Slatkin 1997, Sakal et al 2001). In China (Dong et al 2010) The chosen primers an average of 124. bands, since they generated a total of 498 bands, which ranged from 150-2500bp and qualifies them for barcoding, as seen in table 1. Among the 140 individuals, 435 bands were polymorphic which was different for each primer. In table 2, Neis gene diversity (H) varied between 0. 2612 and 0. 3340, with an average of 0. 3044, and arranged in a descending order the populations, LB KM NN FZ TZ GZ YY while the Shannons information index (I) ranged from 0. 3910 to 0. 4856, with an average of 0. 4499.At the species level, the values of Neis and Shannons showed the same trend as that of PPB. AMOVA analysis showed that there are highly significant (P 0. 001) genetic differences among the seven populations of P. canaliculata. The genetic diversity was by and large due to the differences within the population (92. 76%) while the rest was due t o among populations. The analysis tells the same as that of the Neis and Shannons information, which says that there was a relatively high level of genetic differentiation among populations. CONCLUSIONGenetics of different species are examine by means of DNA barcoding, mostly of the COI gene in the mitochondria, to know the taxon of a species and to understand their trends and characteristics not only morphologically but also genetically. The diversity of a species can also be tested by means of DNA barcoding as seen in the study of the Pomacea canaliculata. The P. canaliculata was introduced in Asia for agricultural purposes and was seen for its benefits but not its drawbacks, which later on resulted to it being invasive and a pest for both humans and other species.To understand the P. canaliculata further, its diversity was studied by means of DNA barcoding and was analyzed both qualitatively and quantitatively in the Philippines and China, respectively. twain the analysis showe d the same outcome, wherein the results showed high levels of genetic diversity among populations. 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