Understanding the degree of genetic variation and how threatened species preserve their genetic diversity is crucial. This knowledge aids scientists in thinking of solutions to prevent the death of species from small populations. Small populations of organisms have a higher risk of going extinct when their genetic diversity is lost since doing so impairs their ability to reproduce and makes them more vulnerable to disease. Research has shown that the arrangement of neutral genetic variation in organisms sometimes fails to show a relationship with the quantifiable variation for traits that determine the ecological fitness of organisms or the variation of genes that are important for the adaptability of organisms (Knopp et al. 50).
MHC Genes: Indicators of Adaptive Capabilities in Organisms
The MHC genes have been relied upon as indicators of adaptive capabilities in organisms in different researches. The genes (MHC) represent the most polymorphic genes in animals with a backbone, which create coded data from a series of glycoproteins that come up with an enhanced organism that can adapt to different ecosystems and be immune to existence-threatening illnesses (Piertney and Oliver 7).
Population Decline of S. chinensis (The Chinese White Dolphin)
S. chinensis is known as the Chinese white dolphin and was mostly found in the coastal and inshore water bodies in the Indian and Western Pacific Oceans. Over time, this dolphin species population has declined considerably. It is listed as one of the organisms that need protection from extinction in the First Order of the National Key Protected Wild Aquatic Animals List in China (Reeves et al. 4). The species has also received international protection due to the declining numbers of its population. Today, the most numerous populations of the species is found in the Pearl River Estuary and consists of about one thousand five hundred individuals (Jefferson and Hung 117). All other areas that have the S. chinensis population make up about three-hundred individuals of the organism.
Genetic Diversity and MHC Gene Expression in S. chinensis
Previous studies have reported the declining genetic diversity among humpback dolphins in the areas. For instance, Lin et al. (416) hypothesized that the consistent bottleneck in the glaciations cycle erode gene load among the dolphins. However, Lin’s (416) hypothesis was based on the evolutionary history of the coastline and not the estuary. The limited genetic diversity has also been attributed to current population contraction caused by the intense anthropogenic factors, including chemical pollution, depletion of prey, and unsustainable ecotourism. Although numerous studies have been conducted on genetic diversity of S. chinensis genes, there have been limited studies about the MCH diversity and MHC gene expression. However, previous studies on different cetacean species have supported the existence of balancing selection on various marine mammals. The present study sought to investigate the expression and diversity of two specific MHC genes (DQB and DRB) in the Chinese white dolphins in the Pearl River Estuary. This study carefully evaluated the basic genetic factors that contributed to the differentiation of MHC gene in S. chinensis. The ultimate goal was to come up with a more effective means of conserving S. chinensis. In the study, the expression and sequence polymorphism of two Major Histocompatibility Complex class II genes (DQB and DRB) of thirty-two Sousa chinensis from the Pearl River Estuary were investigated. The investigation indicated a high ratio of synonymous and non-synonymous substitution rates as well as codon-based selection analysis and TSP (trans-species polymorphism), which supported the hypothesis that Sousa chinensis are subject to a mechanism of balancing selection.
Materials and Methods
Sample Origin, DNA and RNA Isolation, and cDNA Synthesis
Skin samples, as well as muscle samples, were taken from individual dolphins in the Pearl River Estuary. Of the 32 samples taken in this region, no significant variation of the genetic make-up was fond in these organisms. Also, samples from dead individuals were taken such as blood, bodily fluids, and other samples from stranded dolphins. The samples were preserved for research at Sun Yat-sen University. Standard proteinase digestion and phenol-choloform techniques were applied to extract the genomic DNA. RNAiso Plus was used in obtaining total RNA from the samples. The extracted RNA was preserved at -80C while cDNA was build using ologo primers and RNase H Minus reverse transcriptase enzyme. <\p>
PCR Amplification, Cloning, and Sequencing
The research relied on the amplification of the exon number 2 of the DQB and DRB loci, which were collected from the 32 individuals of S. chinensis sample DNA. Using the recognized parameters, a 172 bp section of the exon 2 of the DBQ gene was gauged on the DQF (5′-CATGTGTTACTTCACCAACGGC-3′) and DQR (5′-CACAACT ACAGGRTTGATGAGA-3′) and the results were taken down. In the same way, a 215 bp section of the exon 2 of the DRB gene was amplified with the universal parameter DRR (5′-CCGCTGCA CCGTTGAAGCT-3′), as well as an inverse parameter DRF (5′-CAGTTTAAGKSCGAGTGTC-3′). <\p>
Allele Identification and Nomenclature
Kennedy et al. (348) formulated a criterion that was being used to identify any new alleles. This study followed the criteria established, which needed that a minimum of 3 clones that were identical be present in 2 individual PCR’s from one sample individual, or from 2 PCRs from different individuals during the DNA cloning and sequencing. <\p>
Data Analysis
The results obtained were tested using specialized software to avoid errors that could be caused by the recombinant series in the data. The software analyzed the data statistically to produce a calculated conclusion on the presence and the features of the recombination. Clustal X program was used to align all the sequences. Arlequin 3.5 was applied in calculating the recorded and expected heterozygosities. GENEPOP 4.0 was used to test Hardy-Weinberg equilibrium deviations. Additionally, haplotype and neclotide diversity were calculated by DnaSP 5.0. The presence of DQB and DRB sequences were tested through phylogenetic analysis to remove intralocus recombination. In order to assess whether there was positive selection on the evolution of DQB and D genes, three approaches were used. The first approach involved the calculation of the non-synonymous and synonymous substitution rates for PBR and non-PBR regions. Z-test was used to determine the significance levels. Secondly, CODEML subroutine was used to assess the presence of positive selection at the codon level. <\p>
Data Archiving
To comply with the regulations regarding archiving, the primary data was stored using Dryad. <\p>
Results
Total RNA was extracted from the sample individual with no deletions or insertions. MHC genes were confirmed through the amplification process on the exons 1 to 4 using the primer pairs SoDQ1/2 and SoDR1/2. The loci were identified to be heterozygous and there was no stop codon or deletions/insertions. <\p>
MHC Variability
In terms of MHC variability, sections covering segments of exon 2 of DQB and DRB were amplified successfully from 32 individual. The detected sequences did not have insertions or deletions indicating that the sequence represents functional molecules. According to the analysis barely 2 sequences were detected with each genome indicating that amplification occurred in just 1 DQB locus and 1 DRB locus. <\p>
Selection Analysis
The study did not report any evidence of recombination. DQB and DRB sequences reported increased non-synonymous substitution compared to synonymous rates of substitution. Z-test indicated higher ratio for PBR region thus indicating a positive selection took place at the loci. Based on the CODEML test, the positive selection acted on both the DQB and DRB loci. <\p>
Phylogenetic Analysis
The most ideal model for the DQB data and K12+G data was identified as HKY +G. A phylogenetic reconstruction of the data established clearly supported cetacean-specific clades braches unique from HLA-DQB outgroup, and Caae-DRB sequences. DQB alleles’ phylogenetic analysis indicates TSP similar to those identified in S. chinensis and T. truncates. The phylogenetic analysis indicate the existence of trans-species and trans-genus allelic variations. <\p>
Discussion
In many studies on the genetics of organisms, marine mammals have shown a relatively low level of MHC compared to their terrestrial counterparts (Murray and White 245). In the Chinese white dolphins, the MHC genes depicted an interrupted reading frame which illustrated a deficiency in the MHC function or the result of weakened pathogenic prevalence in the oceanic ecosystem. From the study, the S. chinensis showed a low variation in the MHC class II genes. Both DBQ and DRB alleles were as low in S. chinensis as it is in the P. sinus of California harbor. The P. sinus numbers have been declining globally and stood at slightly over 567 individuals today. The study described successfully DQB and DRB expression through the amplification of exons 1, 2,3,4, from the cDNA which excluded the possibility of pseudogenes amplification. After the cloning of the amplified fragment, cDNA and gDNA reported less than 2 sequences/individuals at every locus. The result suggests the presence of 1 locus for every gene in the S. chinensis genome. <\p>
Low MHC Variation in S. Chinensis
The present study described the differences in the MHC class II gene in the Chinese white dolphin found on the Pearl River Estuary in China. The study was the initial report on the MHC differences. This research established that only the 2 DBQ and 2 DRB alleles could be observed using the methodology and process of accepting new alleles. This observation revealed that there is a significantly low level of MHC class II genes variation in the Chinese white dolphin.<\p>
The Effect of Balancing Selection on MHC Variation
Various studies support the assertion that a balancing selection influenced the MHC sequencing in the Chinese white dolphins. Different genetic alignments and the presence of high ratios of Dn/Ds indicate a long-term tenacity of balancing selection. Initial studies showed that marine organisms were less prone to pathogenic inferiority as compared to the terrestrial organisms. However, this study as well as other studies have disproved the hypothesis and have successfully defended the hypothesis that a limited MHC value determines the natural selection in oceanic mammals. <\p>
Trans-Species Polymorphism
The presence of high dN/dS ratios and Tajima’s D value support the postulation that balancing selection acted on the dolphin MHC sequences. Additionally, the hypothesis is supported by the presence of TS which indicate persistent balancing selection. Balancing selection increases the level of heterozygosity and assumes a key role in maintaining high levels of MHC variation. <\p>
Trans-Species Polymorphism
The study reported that the identified identical and homologous sequences in DQB and DRB loci in dolphins and other whales suggested the existence of TSP. Common ancestry partially explains the persistence of allelic lineages and transmission from species to others. In the presence of polymorphism, sequences ought not to be clustered. The study confirms this through phylogenetic evidence that shows sharing of specific alleles among species. <\p>
Conservation Implications
The presence of low levels of the MHC class II gene in S. chinensis renders it highly susceptible to diseases. However, since the MHC gene in S. chinensis displays a propensity to be divergent, the S. chinensis acquires the capability to resist more illness. <\p>
Works Cited
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Kennedy LJ, Ryvar R, Gaskell RM, Addie DD, Willoughby K, Carter SD, Thomson W, Ollier WE, Radford AD. “Sequence Analysis of MHC DRB Alleles in Domestic Cats from the United Kingdom.” Immunogenetics, vol. 54, 2002, pp. 348–352.
Knopp, Theresa, José M. Cano, Pierre-André Crochet, and JuhaMerilä. “Contrasting Levels of Variation in Neutral and Quantitative Genetic Loci on Island Populations of Moor Frogs (Ranaarvalis).” Conservation Genetics, vol. 8, no. 1, 2006, pp. 45-56.
Lin, W. Z., Chang, L. H., Frere, C. H., Zhou, R. L., Chen J. L., Chen, X., and Wu, Y. P. “Differentiated or Not? An Assessment of Current Knowledge of Genetic Structure of Sousa Chinensis in China.” Journal of Experimental Marine Biology and Ecology, vol. 416-417, 2012, pp. 16–20.
Murray, B. W., and White, B. N. “Sequence Variation at the Major Histocompatibility Complex DRB Loci in Beluga (Delphinapterus Leucas) and Narwhal (Monodon Monoceros).” Immunogenetics, vol. 48, 1988, pp. 242–252.
Piertney, S. B., and M. K. Oliver. “The Evolutionary Ecology of the Major Histocompatibility Complex.” Heredity, vol. 96, no. 1, pp. 7-21.
Reeves, R. R., Dalebout, M. L., Jefferson, T. A., Karczmarski, L., Laidre, K., O’Corry-Crowe, G., Rojas Bracho, L., Secchi, E. R., Slooten, E., and Smith, B. D. Sousa Chinensis. IUCN Redlist of Threatened Species, 2010.
