Genetic Polymorphism in Animals

Abstract

Biological diversity is the variability among living organisms from all sources of nature. Genetic polymorphism study support a lot when any economically important particular species is taken into consideration. The knowledge of genetic background of a species and its population structure is very essential for their successful conservation and management. Molecular techniques have been supporting in the determination of population diversity and also to determine the genetic architecture of a wide variety of closely related individuals. Molecular techniques based on DNA polymorphism are now used in population genetic studies, systematic and molecular taxonomy. This chapter will provide information on genetic diversity of various economically important species such as protozoa, worms, insects, pearl oyster, fishes and birds. The study of genetic variations in economically important species has practical significance for developing strategies to control the disease, to improve reproductive traits, yield more beneficiary products like honey, silk, pearl, manure, etc. Since there are some data gaps, most suitable and promising technology must be used to elucidate the role of every single gene involved in the pathways to be studied in order to apply for more benefit to the society.

1. Introduction

Biological diversity is the variability among living organisms from all sources of nature. Information on molecular structure of economically important organism is useful for optimizing identification of stock, stock enhancement, breeding program, management of sustainable yield and preservation of diversity. Genetic polymorphism study plays an important role to understand the basis of population differentiation and species diagnostics. In recent times molecular techniques have been supporting in the determination of population diversity and also to determine the genetic architecture of a wide variety of closely related individuals. The discovery of PCR has a major impact on eukaryotic genome and contributed to the development and application of various DNA markers. Within the population, organisms are identified by their morphology but there are some small invisible changes observed due to environmental effect. Genetic polymorphism study support a lot when any economically important particular species is taken into consideration.

This chapter will provide information on genetic diversity of various economically important species such as Protozoa, Worms, Insects, Pearl oyster, Fishes and Birds.

2. Protozoa

There are many factors associated with protozoan parasites genetic diversity such as: transmission and passage history in laboratory conditions; occurrence in different hosts or geographic regions; selective pressure of drugs and competitive interactions among populations. However, the number of examined isolates of parasites and genetic markers, assortment of methods, probes, primers and reagents used is also of significance. The significance of genetic variability in parasite populations is still the subject of interest and controversy. A simple interpretation of such variation is impossible because of the complexity of host–parasite interactions. The knowledge of parasite diversity at the nucleic acids level has continually increased, but a correct interpretation of this phenomenon requires at least the same knowledge of genetic variability in host populations [1].

The study of genetic variation in malaria parasites has practical significance for developing strategies to control the disease. The genome diversity of the important human pathogen Plasmodium vivax, however, remains essentially unknown. The data about Single Nucleotide Polymorphisms (SNPs) show that P. vivax has a highly diverse genome, and provide useful information for further understanding the genome diversity of the parasite [2].

All RAPD primers yield patterns that differ between species and thus could serve as species-diagnostic traits. But the extensive polymorphisms observed within each species for most RAPD primers preclude their practical use for species diagnosis. There are, however, primers that yield monomorphic patterns within a species and thus can readily be used for species diagnosis, which may be useful for epidemiological and other purposes. For example, two primers are monomorphic in T. cruzi and thus could be used in epidemiological practice for differentiating T. cruzi from T. rangeli, a species that infects humans [3].

3. Worms

For stem cells and regeneration study, free-living flatworms (the planarian Schmidteamediterranea) are extensively used as model organisms. The germline-enriched genes of the flatworm M. lignano have a high fraction of flatworm-specific genes. The Mlig-sperm1 gene responsible for producing healthy spermatozoa has been identified as a member of the novel gene family conserved only in free-living flatworms [4].

TIM29 (mitochondrial inner membrane protein) was shown to stabilize the protein import complex TIM22 by interacting with it, but its biological function remains largely unknown. Till now, it was classified as one of the Domain of Unknown Function (DUF) genes, with a conserved protein domain DUF2366 of unclear function. It has been demonstrated that DUF2366/TIM29 knockdown in Macrostomumlignano prevents worms from adapting to a highly proliferative state required for regeneration with least effect during the normal homeostatic condition [5].

Low response to ivermectin (IVM) in patients infected with Onchocerca volvulus indicates that the parasite might be under a selection process toward potential resistance. In order to limit this process, the characterization of O. volvulus genes is very crucial. It has been observed that a deficit of heterozygous female worms leading to Hardy Weinberg disequilibrium, which might be explained by a shorter life-span of these worms compared to the homozygous worms. Also the heterozygous female worms were much less fertile than the homozygotes: more than two thirds of the homozygotes were fertile, whereas only 37% of the heterozygotes were fertile [6].

Control of onchocerciasis or river blindness by mass treatment of the population with IVM has been a great success until now, so that in certain foci its elimination has become feasible. However, after more than 20 years of repeated IVM mass treatment, the disease still persists in many endemic countries. Sub-optimal responses and genetic changes have been reported in Onchocerca volvulus populations under high IVM pressure but more work is needed to determine whether resistance is developing. In a study four SNPs occurring in the β-tubulin gene of these parasites were investigated and found changes in genotype frequencies in O. volvulus β-tubulin gene associated with IVM treatments. The SNP at position 1545 (A/G) showed a significant increase in frequency of the less common nucleotide in the female worms following treatment. After three-monthly treatments, female worm homozygotes with the less common genotype, prior to treatment, increased in frequency. The selected homozygotes, as well as heterozygotes, appeared to be less fertile than the wild-type homozygotes. These results provide additional evidence for genetic selection [7].

4. Earthworms

From a unicellular organism like protozoans, nature has used its nimble fingers to create the structural complexity inside the organisms at diverse stages, to conform themselves very correctly to the prevailing conditions. In this scheme of their polymorphism, it’d be suitable to emphasize the evolutionary significance of earthworms which cause them to masters the soil invertebrate community. In general, lumbricid earthworms have ruled of their distribution in temperate soils and the megascolecid earthworms predominate the sub-tropical and tropical soils. The depth of competition is intense in temperate areas with a slender area of interest where litter forms the primary food source [8]. On the opposite hand, niche and morphogenecity have been enlarged with greater food diversity for tropical earthworms and they display awhole lot of variant in size and behavioral patterns.

4.1 Molecular markers and earthworm genetic polymorphism

Despite being efficient in soils, their relevance and research over the past 130 years are still fragmentary therefore, the various species complex and morphs are not being resolved adequately. Molecular systematics can be an essential supply of facts to delimit species and to assign taxonomic categories in complex species. Several genes including cytochrome oxidase 1 (COI), 18S, 16S, 28S, and protein-coding histone H3 genes had been currently used for reading phylogeny and polymorphism in earthworm species. The COI gene is a 658 bp trendy marker that has been demonstrated to be powerful for earthworm identifications, molecular systematics, ecology, phylogeography, and cryptic speciation. Furthermore, the other markers specifically 18 s, 28 s, are often involved to have a look at interfamily, intrageneric, and shallow intraspecific diversity. Most of the polymorphic research are primarily based on mitochondrial markers mainly COI, COII, and different protein-coding genes as they evolve extra unexpectedly than nuclear genes thus resulting in the accretion of differences between closely related species.

The Barcode of Life Data System (BOLD, http://www.barcodinglife.org), a valuable integrative bioinformatics platform, serves as a systematic workbench helping all stages of the analytical pathway from specimen collection to validation was formulated by Ratnasingham [9]. Chang et al. [10] investigated polymorphism in Metaphire formosae species group, a member of the Pheretima complex using DNA sequences of (COI), 16S ribosomal (r)RNA, and NADH dehydrogenase subunit 1 (ND1) and revealed the presence of 13 taxa of the M. formosae species group, including a cryptic species. A study sequenced the COI, 16S, tRNAs, and 28S genes in 202 Hormogastridae earthworm individuals in the Iberian Peninsula and suggested the presence of excessive genetic range with the presence of five cryptic allopatric species [11]. Another study identified two new earthworm species namely E. nordenskioldimongol and E. nordenskioldionon from Mongolia which were justified by molecular taxonomy using mitochondrial DNA barcoding [12]. Similarly, Dario et al. [13] provided the description of one new earthworm species viz., Eiseniona gerardoi primarily based on mitochondrial in addition to nuclear molecular markers, within the controversial genus Eiseniona of lumbricidae from the region of Extremadura (Spain). Dominguez et al. [14] studied the evolution of lumbricids via way of means of reading one hundred sixty earthworm individuals using the sequencing of two nuclear genes and seven mitochondrial tRNAs, with 22 morphological characters, discovered 84 lumbricid species under 28 genera. Also, Dmitry and Gennady [15] studied the taxonomical status of the exceptional ecological forms of D. ghilarovi using COI and 16 s genes as molecular tools. They discovered apparent differentiation among the meadow-swamp black form and the forest gray form of D. ghilarovi. Decaens et al. [16] sequenced 651 individuals using COI gene, which corresponds to 48 MOUTs, and concluded that factors like ecological processes in addition to long-time period diversification are critical in structuring and diversity of earthworm communities in tropical rainforests of French Guiana. Moreover, Hong and Csuzdi [17] revoked the phylogenetic affinities of Korean E. nordenskioldi specimens and compared them with Siberian E. nordenskioldi individuals on the basis of mitochondrial gene marker. Also, Shekhovtsov et al. [18] investigated the genetic variations of Eisenia norden skioldipallida Malevic from various climatic zones of Northern Asia by sequencing of COI and ITS2 loci and detected five cryptic genetic lineages within E. nordenskioldipallida. Teerapong et al. [19] explored new earthworm species, Pontodrilus longissimus sp. nov., from beaches of Thailand and Peninsular Malaysia primarily based on morphological investigations and partial sequencing of mitochondrial COI gene. The molecular phylogeny of Lumbricidae earthworms was investigated by Farnaz et al. [20] using phylogenetic evaluation of two nuclear gene regions (28S rDNA and 18S rDNA) and 11 mitochondrial genes (16S rDNA, 12S rDNA, NADH-I, COI and COII and tRNAsAsn, Asp, Val, Leu, Ala and Ser) that lead to the addition of one new genus; Philomontanus gen. Nov and its three new species namely, Philomontanussarii sp. nov., Philomontanusmahmoudi sp. nov. and Philomontanusbaloutchi sp. nov.

In India, the studies on earthworm polymorphism using molecular tools were unavailable till 2008. However, two studies used the RAPD-PCR technique for the first time to evaluate the genetic diversity of Indian earthworms [21, 22]. Various molecular markers viz., Random Amplified Polymorphic DNA (RAPD), Restriction Fragment Length Polymorphism (RFLP), and Simple Sequence Repeat (SSR) [23, 24] were sequenced in addition to mitochondrial COI gene of six species of earthworms and determined that the COI exhibited a unique barcode to a particular species. One of the latest works [25] explored four new species of Kanchuria (Megascolecidae) from Meghalaya and updated the checklist of the Northeastern region of India [26]. Also, Thakur et al. [27] studied polymorphism in Eutyphoeus sp. using mitochondrial gene marker. Tiwari et al. [26] studied the earthworm polymorphism of the Sagar district of India using integrative methods. Nonetheless, earthworm molecular taxonomy based on DNA sequencing in India is at its initial stage, such that very little information is available. Recently, 801 DNA sequences of Indian earthworm (belonging to family Acanthodrilidae 4, Almidae 3, Eudrilidae 22, Hormogastridae 7, Lumbricidae 6, Megascolecidae 426, Moniligastridae 122, Octochaetidae 199, Rhinodrilidae 6) have been made available on Bold-system under the project diversity studies of Indian earthworms using DNA barcodes. This signifies only a fraction of Indian earthworms and thus further challenges remain open for biology fanatics to research in the field of genetic polymorphism using molecular approach.

4.2 Reproductive organs polymorphism

The absence of certain reproductive organs is ordinarily essential for biparental replica in hermaphroditic oligochaetes [28]. During the evolutionary process, the organs important in biparental reproduction in hermaphroditic animals were found removed in certain Japanese species of Pheretima and evolved into a various intraspecific morphs viz., H (primary, from which different morphs derived and with biparental replica), Hp (secondary, reproductive machine incomplete however a number of organs stay juvenile), A (without spermathecae, storage of sperm impossible), R (without terminalia, discharge of sperm impossible), AR (without spermathecae and terminali, storage of foreign sperm and extrusion of own sperm impossible) and ARZ (testes and other male organs and spermathecae lacking). That reproductive-organ polymorphism in Pheretima emphasizes a normal alternate into being uniparental. Organ disorder denial of alternative among uni and biparental may also make parthenogenesis pseudo-obligatory in selected complex of earthworms.

4.3 Biochemical polymorphism in earthworms

Since, the genetic variation in earthworm species may cause mild effect on phenotype, voluminous work is focused on the effects of only one or a few polymorphic loci at any one time. There is an upcoming concern of an individual possessing numerous polymorphism (set of polymorphic loci), which may act in concert to affect phenotype. That can be assessed through correlating genetic houses of loci with physical and/or biochemical properties of the respective gene products. The variation in distribution of polymorphism among different pathways such as glycolysis, Kreb’s cycle, hexose monophosphate shunt inside glucose metabolism and ancillary pathways leading to amino acid metabolism was first highlighted [29]. The polymorphic loci which persist in conserved pathways should have common variants with intermediate biochemical activities and less uncommon alleles than polymorphic loci in different pathways. Comparisons of the activities of rate limiting enzymes among different invertebrates suggest that earthworms have a fairly low metabolic rate [30, 31, 32]. The studies are beneficial particularly in resolving polytypic complexes and the genetic consequences of parthenogenesis in invasive species.

5. Insects

Insects represent a major life form on earth. So far nearly 0.9 million insect species have been discovered comprising 75% of all the recorded animal species. Some of the insect species are easy to identify and categorize while for others it is difficult due to their small size and environmental factors that resulted in to morphological variation. To overcome these problems, the advanced molecular techniques viz. PCR, RFLP, and ALFP have been a great help. RAPD markers have been used in gene mapping to characterize cultivars and species genetically, infer phylogeny and biogeography of insect population and understand modes of evolution and evolutionary trajectories [33]. RAPD-PCR analysis was used to confirm genetic differentiation between two cockroach species Perplaneta Americana and Blatellagermanicana [34].

5.1 Honey bee

Honey bee is the colonial insect with complex social behavior. Beside its economic importance it has long been important for the production of honey, wax, behavioral study and pollination of crops. Nemobiologists and behaviorists have used the honey bee as a model organism to study the molecular basis of learning [35]. For genetic analysis honey bee genome sequencing project also proposed that it will benefit human health and medicine in diverse areas including venom toxicology, allergic diseases, mental illness, infectious diseases, parasitology and gerontology and will also improve human nutrition by enabling enhanced pollination of food plants and accelerated delivery of hymenopteran parasitoids for biological control of pests [36]. Honey bee has a higher rate of meiotic recombination than any other known metazoan [37]. The higher recombination rates effectively increase the accuracy of linkage mapping and high recombination rate and the low incidence of repetitive DNA should facilitate map based cloning of genes in the honey bee [38]. AFLP markers and microsatellites have been used in dissecting the guarding and stinging behavior in honey bee. Division of labour, expression of guarding and stinging behavior is influenced by specific quantitative trait loci [39]. By using multilocus fingerprinting super and half-sister in a colony of honey bee has been discriminated with synthetic oligonucleotide [40].

5.2 Silkworm

The silkworm Bombyxmori is domesticated for silk production for about 5000 The silkworm B. mori is domesticated for silk production for about 5000 years. The well characterized mutations of B. mori (a well studied lepidopteran model system) affect every aspect of organism’s morphology, development, behavior and its considerable economic importance [41].

Since B. mori is of great economic importance to silk producing countries such as India, Russia, Japan, Korea, China, Bulgaria and Iran, a number of silkworm breeds have been collected suitable for a wide range of agroclimatic conditions. More than 4000 strains are maintained in the germplasm of B. mori and 46 institutes are involving silkworm genetic resources maintenance, which includes univoltine, bivoltine, and polyvoltine strains. These different genotypes possess so many differences in their quantitative and qualitative traits which control silk yield. It was estimated that silkworm genome contains about 108 bp and its genetic information volume is nearly one-sixth of human being. There are over 450 morphological, physiological, and biochemical characters recorded at present, among them 300 (including multiallele) had been located on 27 groups of the total 28 chromosomes [41]. Apart from a rich biodiversity of geographical races, there are also a large number of mutants for a variety of characters present in B. mori [42]. Zhang et al. [43] reported that genetic distances within Japanese strains are closer than those of Chinese strains and within a strain; the individual polymorphism is significantly higher in wild silkworm than those of domesticated silkworm. According to Liu [44] at the species level, Antheraeapernyi and Bombyxmori showed high levels of genetic diversity, whereas Samia Cynthia ricini showed low level of genetic diversity. However, at the strains level, Antheraeapernyi had relatively the highest genetic diversity and B. mori had the lowest genetic diversity. Yukuhiro et al. [45] analyzed PCR amplified carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase (CAD) gene fragments from 146 Bombyxmori native strains and found extremely low levels of DNA polymorphism. CAD haplotype analysis of 42 samples of Japanese B. mandarina revealed four haplotypes. No common haplotype was shared between the two species and at least five base substitutions were detected. These results suggesting that low levels of gene flow between the two species. Further extremely low level of DNA polymorphism in B. mori compared to its wild relatives suggested that the CAD gene itself or its tightly linked regions are possible targets for silkworm domestication. This information clearly indicates narrow level of genetic diversity in silkworm.

The existence of genetic variation within a population is crucial for its ability to evolve in response to novel environmental challenges. In order to adapt to new environment and conditions, the genetically variable populations are thought to evolve their morphological, physiological or behavioral mechanisms [46]. This not only results in better adaptation to their local environments, but may also lead to reduction in the genetic variations thereotically. Domestication, phenotypic selection, breeding systems, genetic drift is thought to be the main reason for reduced genetic diversity in silkworms.

6. Pearl oyster

The pearl oyster Pinctadafucata is a commercially important marine shellfish cultured for producing saltwater pearls manly in China and Japan [47]. Researchers using genome-wide genetic data from specimens collected across the western Pacific, elucidated how pearl oyster populations vary genetically and geographically. Their analyses provide insight into how these pearl oysters have adapted to environmental changes over time. They sequenced the genomes of the specimens and analyzed 36,203 single nucleotide polymorphism (SNP) sites [48].

Genetic variability and the pattern of population structure among 9 samples of Calafia pearl oyster Pinctadamazatlanica collected from Mexico to Panama, using mtDNA RFLP analysis of two genes 12S rRNA and subunit one of Cytochrome oxydase (COI). Haplotype diversity varied from 0.000to 0.856. The Panama population appeared to be monomorphic, while the other samples exhibited a level of haplotypic variability. A test for the impact of demographic history on genetic diversity was applied on the sequence data, and the results were congruent with a recent decline of population sizes. Three significantly distinct groups could then be defined, which correspond to Northern Mexico, Southern Mexico, and Panama [49]. The pearl oyster, Pinctadafucata, a marine bivalve belonging to the family Pteriidae, is the primary species cultured for marine pearls in China and Japan [50]. In a study 16 SNP markers have been reported, may provide a useful tool for population genetics and evolutionary analysis [51].

7. Fish

Fishes are economically important animal consumed globally. Information on the molecular structure of fish species is useful for optimizing identification of stocks, stock enhancement, breeding programs, and management of sustainable yield and preservation of genetic diversity. The knowledge of genetic background of a species and its population structure is very essential for successful fisheries conservation and management. A number of methods have been developed to measure genetic diversity within the species [52]. Molecular techniques based on DNA polymorphism are now used in population genetic studies, systematic and molecular taxonomy. Molecular techniques played an important role to understand the basis of polymorphism of a species, species diagnostics and population differentiation. Three fish species of fish Labeorohita, Catlacatla, Cirrhinamrigala (family Cyprinidae) of India have beenstudied by RAPD for molecular identification. The diverse nature of DNA bands indicated the genetic distance between fish species and presence of common bands attributed to an evolutionary relationship. Pattern of species specific unique bands are useful for identification [53]. In addition to Labeorohita and Catlacatla, another fish Nile tilapia of the same family has also been studied showing genetic relationship [54]. Kempter et al. used microsatellites for the authentication of torpedo scad (Megalaspiscordyla) -fish species by successfully extracting a DNA fragment of the nuclear rhodopsin gene (RH1) and amplification of nine microsatellite regions (SSRs). After analysis of the products, they found differences between the RH1 sequences and those obtained from Gene Bank. This study was used to characterize and assess the genetic diversity of the populations as well as effectively categorized the populations over the areas of the western Indian Ocean and the western Pacific [55].

8. Birds

Major factors of economic concern in the modern poultry industry are the reproductive traits i.e. age at first egg, number of eggs and weight of the egg [56, 57, 58, 59]. These reproductive traits are of great importance as studies on these genes helps in revealing the genetic mechanisms affecting egg-laying performance and for breeding the laying hens with high productivity and quality [60, 61, 62, 63, 64]. RT-PCR results of a study showed that the growth differentiation factor 9 (GDF9 gene) is involved in determining reproductive traits in chicken as they observed its high expression in stroma with cortical follicles (STR) and prehierarchal follicles [62].

Very low density apolipoprotein-II (apoVLDL-II) is a major polypeptide component of avian VLDL. The function of apoVLDL-II is the transport of neutral lipids (triacylglycerol) in the form of VLDL in the plasma. The apoVLDLII gene is dormant in embryos, chicks and roosters but can be activated by estrogen. Genotyping for the apoVLDL-II gene showed a mutation in 492-bp fragment located on the first intron. Polymorphism in apoVLDL-II gene was significantly associated with body weight at 6 week (BW6), carcass weight (CW), breast muscle weight (BMW), drumstick weight (DW) and wing weight (WINW). Association between single nucleotide polymorphism of apoVLDL-II gene with growth and body composition traits in Iranian commercial broiler line [65].

Genetic diversity measured at the molecular level does not always correspond to phenotypic breed diversity, because a long history of exchange, upgrading and crossbreeding has sometimes created new genotypes within old phenotypes. For example, breeders of fancy breeds are mainly concerned about the phenotype, whereas the genotype of phenotypically different breeds may be very similar [66].

In a study, it has been reported that the three structurally related orphan G protein-coupled receptors (GRP3, GPR6, GPR12) are constitutively active and thought to regulate neuronal outgrowth and oocyte meiotic arrest in mammals. But the information is scanty related to this data in case of non mammalian vertebrates therefore require further research. The cloned duck GPR3 and duck/chicken GPR6 and GPR12 are intron-less and encode receptors that show high amino acid sequence identities (66–88%) with their respective mammalian orthologs. It has been demonstrated that GPR3, GPR6, GPR12, and GPR12L are constitutively active and capable of stimulating the cAMP/PKA signaling pathway without ligand stimulation in birds (and zebrafish), indicating their conserved signaling property across vertebrates [67]. Several researches have demonstrated that RNA-Seq is the most suitable and promising technology which gives a definitive view of the pathways involved and the role of every single gene in that process [68]. RNA-seq data/qRT-PCR assays revealed that GPR6 and GPR12L expression is mainly restricted to the chicken brain, while GPR12 is highly expressed in chicken ovarian granulosa cells (GCs) and oocytes of 6 mm growing follicles and its expression in cultured GCs is upregulated by progesterone [67].

Signal transducers and activators of transcription (STATs) represent a family of latent cytoplasmic proteins that mediate a variety of peptide hormones and cytokines in a target cell [69], controlling the action of growth hormones on target genes [70]. STAT5B regulates ovary development and sexual maturation [71]. Moreover, in another study, STAT5B knock-out mice had no breast development or lactation [72].

9. Conclusion

The study of genetic variations in economically important species has practical significance for developing strategies to control the disease, to improve reproductive traits, yield more beneficiary products like honey, silk, pearl, manure, etc. Further, the study of genetic variation will help in area wise differentiation of the species. Also, the data pertaining to G protein-coupled receptors (GRP3, GPR6, GPR12) is scanty in case of non mammalian vertebrates which needs further research. RNA-Seq -the most suitable and promising technology must be used to elucidate the role of every single gene involved in the pathways to be studied in order to apply for more benefit to the society.