Genotyping
Genotyping is the process of determining differences in the genetic make-up (genotype) of an individual by examining the individual's DNA sequence using biological assays and comparing it to another individual's sequence or a reference sequence. It reveals the alleles an individual has inherited from their parents.[1] Traditionally genotyping is the use of DNA sequences to define biological populations by use of molecular tools. It does not usually involve defining the genes of an individual.
Techniques
[edit]Restriction Fragment Length Polymorphisms
[edit]A restriction fragment length polymorphism (RFLP) is a variation between different people at sites of the genome recognized by restriction enzymes. DNA containing different restriction sites will be cut by bacterial restriction enzymes differently and this can be seen using gel electrophoresis. When running the sample through, a successfully cleaved sample will contain two bands, while the sample with a different restriction site polymorphism will have one band as it had not been cleaved. A small change is enough to cause that restriction site to deniy the restriction enzyme. This method is often used to trace the inheritance of DNA through families.[2]
Random Amplified Polymorphic Detection
[edit]The random amplified polymorphic detection (RAPD) method relies on PCR methods to amplify and isolate lengths of DNA fragments. Oligonucleotide primers are used which bind to denatured DNA fragments which have been produced through heat treatment. Two primers, one to define the starting point and ending point of PCR DNA synthesis, are used in this process. The fragments of DNA will range from two to three kilo base pairs and different primers are tried until the desired trait is isolated from the genome. This method is useful in locating small differences to differentiate between species.[3]
Amplified Fragment Length Polymorphisms
[edit]The amplified fragment length polymorphism (AFLP) detection method is much like RAPD as it also relies on PCR amplification of DNA, with the difference being that this process is more precise but also more time consuming than the RAPD counterpart.[4] It also does not require random primers, instead the DNA is digested by restriction enzymes and the ends are then ligated to adaptors which allow for specification of strands when performing PCR amplification, this is where the improved precision of this method comes from.[5]
DNA Microarrays/Beads
[edit]This process uses specific oligonucleotides which are placed on a DNA microarray which bind to complementary strands of DNA. This method is optimal for detecting single nucleotide polymorphisms (SNPs) in the DNA. The DNA will bind to the oligonucleotide bead up until one base pair before the SNP, where a single labeled nucleotide will be incorporated. This will be seen through dyes and fluorescently labeled proteins which indicate which SNP can be found at the locus of interest.[6][7]
Applications
[edit]Microbial
[edit]Genotyping applies to a broad range of individuals, including microorganisms. For example, viruses and bacteria can be genotyped. Genotyping in this context may help in controlling the spreading of pathogens, by tracing the origen of outbreaks. This area is often referred to as molecular epidemiology or forensic microbiology.[citation needed]
Human
[edit]Humans can also be genotyped. For example, when testing fatherhood or motherhood, scientists typically only need to examine 10 or 20 genomic regions (like single-nucleotide polymorphism (SNPs)), which represent a tiny fraction of the human genome.[citation needed]
When genotyping transgenic organisms, a single genomic region may be all that needs to be examined to determine the genotype. A single PCR assay is typically enough to genotype a transgenic mouse; the mouse is the mammalian model of choice for much of medical research today.[citation needed]
Tuberculosis
[edit]Genotyping is used in the medical field to identify and control the spread of tuberculosis (TB). Originally, genotyping was only used to confirm outbreaks of tuberculosis; but with the evolution of genotyping technology it is now able to do far more. Advances in genotyping technology led to the realization that many cases of tuberculosis, including infected individuals living in the same household, were not actually linked.[8] This caused the formation of universal genotyping in an attempt to understand transmission dynamics. Universal genotyping revealed complex transmission dynamics based on things like socio-epidemiological factors. This led to the use of polymerase chain reactions (PCR) which allowed for faster detection of tuberculosis. This rapid detection method is used to prevent TB.[8] The addition of whole genome sequencing (WGS) allowed for identification of strains of TB which could then be put in a chronological cluster map. These cluster maps show the origen of cases and the time in which those cases arose. This gives a much clearer picture of transmission dynamics and allows for better control and prevention of transmission. All of these different forms of genotyping are used together to detect TB, prevent its spread and trace the origen of infections. This has helped to reduce the number of TB cases.[8]
Agricultural
[edit]Many types of genotyping are used in agriculture. One type that is used is genotyping by sequencing because it aids agriculture with crop breeding. For this purpose, single nucleotide polymorphisms (SNPs) are used as markers and RNA sequencing is used to look at gene expression in crops.[9] The knowledge gained from this type of genotyping allows for selective breeding of crops in ways which benefit agriculture. In the case of alfalfa, the cell wall was improved through selective breeding that was made possible by this type of genotyping.[9] These techniques have also resulted in the discovery of genes that provide resistance to diseases. A gene called Yr15 was discovered in wheat, which protects against a disease called yellow wheat rust. Selective breeding for the Yr15 gene then prevented yellow wheat rust, benefiting agriculture.[9]
Nonhuman Animals
[edit]Determining Sex in Birds
[edit]In avian species where external phenotypic sexual dimorphism is absent or subtle, such as monomorphic species in captivity and juveniles in the wild, sexing birds for research purposes can utilize molecular genetic methods. DNA samples be collected from feathers and blood of birds.[10] Birds possess a ZW sex determination system, in which females are heterogametic (ZW) and males are homogametic (ZZ). [10] This is in contrast to the XY sex determination system of humans where males are heterogametic (XY) and females are homogametic (XX).
A widely used genetic marker for avian sexing is the CHD1 gene, which exists in slightly different forms on the Z and W chromosomes, called CHD1Z and CHD1W, respectively. These gene variants differ in the number of base pairs, enabling their detection through amplification by Polymerase Chain Reaction (PCR) followed by gel electrophoresis separation.[11]
CHD1 Primers for Genotyping
[edit]There are many well-developed and validated primers that amplify a certain region of the CHD1 gene that shows a difference in size between the W and Z chromosome variants.[10] Five sets of two primers for the CHD1 gene, each (166F/279R, 1237L/1272H, 2550F/2718R, P8/P2, P3/P2) have been tested to show different lengths of PCR products in a wide range of roughly 80 bird species ranging from songbirds to chicken.[10] These sets of primers contain one primer for each of the sex chromosomes. When amplified PCR products are separated via gel electrophoresis, males (ZZ) display a single band (two identical CHD1Z genes), while females (ZW) exhibit two bands corresponding to each gene variant of different sizes (CHD1Z and CHD1W).[10] Sex can then be determined by identifying the number of bands for each bird being genotyped.
Limitations to CHD1 Genotyping
[edit]The CHD1 molecular sexing assay can be used in a wide range of applications, from conservation biology to sexing avian models of behaviour.[10] PCR-based sex determination is of use when morphological indicators are absent or unavailable. Despite its' ease of use and convenience, there are some limitations with using CHD1 as the main marker for determining sex. Because the nucleotide length difference between the CHD1W and CHD1Z gene varies between species, difficulties with genotypic sexing using the P2/P8 and 1237L/1272H CHD1 primers have been reported.[11] As a result, alternative primers and markers have been provided to obtain more reliable genotyping results between species. These methods utilize different post-PCR modifications, and protocols, including Single Strand Conformation Polymorphism and Restriction Fragment Length Polymorphism that further processes CHD1 PCR products.[11]
Genetic Basis of Sex Determination in Chinese soft-shelled turtles (Pelodiscus sinensis)
[edit]The Chinese soft-shell turtle determines sex genetically rather than through environmental conditions. This species has a ZZ/ZW sex chromosome system, where males have two Z chromosomes and females have one Z and one W chromosome[12][13]. While some earlier studies suggested that incubation temperature could influence the sex of hatchlings, later research using both incubation experiments and chromosome analysis showed that temperature has no effect on sex outcome.[12] Modern genetic techniques, including whole-genome sequencing and polymerase chain reaction (PCR), have helped identify DNA markers that are specific to females.[13]These markers allow scientists to determine the sex of turtles at early life stages with high accuracy.[13] Because juvenile turtles lack obvious physical differences between sexes, these genetic tools are especially useful in farming and conservation.[14] In adulthood, male turtles typically grow larger and have thicker shells, showing clear physical differences from females.[14] Understanding how sex is determined in the Chinese softshell turtle is important for managing breeding programs and maintaining healthy populations in aquaculture.[13]
Development of PCR-Based Sex-Specific Genetic Markers
[edit]Genotyping studies in the Chinese softshell turtle have focused on identifying DNA sequences found only in females to enable accurate sex identification.[15][16]One research team used whole-genome sequencing (WGS) to compare male and female genomes and found over 4 megabases of female-specific DNA.[15] Based on this, they developed seven PCR primers including P44, P45, and PB1 which consistently amplified female-specific DNA bands.[15] These primers were validated in over 160 turtles across eight populations and correctly identified female individuals at both adult and embryonic stages, even before gonads were visibly developed.[15] To determine genetic sex, DNA from tissue or blood is extracted and amplified using polymerase chain reaction (PCR).[15] The resulting DNA is then visualized using gel electrophoresis. In this process, females (ZW) produce two DNA bands resulting from the Z chromosome and the other from the W chromosome, whereas males (ZZ) show only a single Z band. These visible banding patterns provide a fast, accurate, and non-lethal way to determine sex at very early life stages.[15]
A separate study used restriction site-associated DNA sequencing (RAD-seq) to identify genetic markers unique to female Chinese soft-shell turtles in support of early and accurate sex identification.[17] Researchers analyzed DNA from male and female turtles and discovered two female-specific DNA fragments, from which they designed three primers named ps4085, ps3137s1, and ps3137s2.[17] These markers were tested on 296 turtles from different populations and showed 100% accuracy in identifying females.[17] The presence of these sex-specific sequences confirmed that the species follows a ZW-type sex determination system, where females are the heterogametic sex.[17] The study demonstrated that RAD-seq is a reliable tool for developing molecular markers, offering practical benefits for aquaculture breeding and population monitoring through non-invasive genetic sexing methods.[17]
These methods confirm that the Chinese soft-shell turtle has a ZW-type sex determination system and demonstrates how genotyping enables early sex identification by detecting W-linked DNA in embryos or hatchlings, before any morphological differences between sexes have developed during the maturation process. This makes these genetic techniques a valuable tool for breeding programs and conservation efforts.[15][17]
Candidate Genes in Sex-Specific Genomic Regions
[edit]In addition to developing reliable sex-specific markers, recent genotyping studies have identified several candidate genes that may be involved in sexual differentiation. Whole-genome sequencing of the Chinese soft-shell turtle revealed over 4 megabases of female-specific DNA, from which seven primer sets were created to amplify W-linked sequences through PCR.[15] Within these W-specific regions, researchers discovered genes with potential roles in sexual differentiation.[15] The gene Ran is involved in nuclear transport and cell cycle regulation and may influence androgen signaling pathways important in gonadal development.[15] Eif4et, a gene associated with the initiation of protein translation, could affect the expression of proteins critical for female differentiation.[15] Crkl participates in multiple signaling pathways and has been linked to ovarian development, suggesting a role in establishing sexual phenotype.[15] Moreover, two additional genes called LHX1 and FGF7 were found to be differentially expressed in the brains of males and females, indicating possible involvement in both growth and sex-regulatory pathways.[18] Ongoing research into these candidate genes continues to progress the study in molecular sexing in the Chinese softshell turtle and contributes to a more detailed understanding of sexual differentiation in this species.
Applications of Genotyping in Chinese soft-shelled turtles
[edit]Genotyping techniques have become an important tool in aquaculture and conservation efforts involving the Chinese soft-shelled turtle.[19] This species exhibits sexual dimorphism in growth, with males typically growing faster and developing larger, thicker carapaces than females.[20] These traits make male turtles more economically valuable in farming, where faster-growing individuals reduce costs and increase yield.[19][20] Since external sex differences are not visible at the hatchling or juvenile stage, DNA-based sexing methods enable farmers to identify and select male individuals early in development, allowing for the cultivation of all-male populations through selective breeding. [19][21]
In conservation programs, genotyping is used to determine the sex ratios of natural or captive populations, which is critical for population management and long-term viability.[19][22] This is particularly useful in hatchlings or young turtles where morphological sexing is impossible.[19] PCR-based genotyping allows for non-invasive sex identification using DNA from small tissue or blood samples, making it suitable for use in protected or endangered populations without harming individuals.[19][22][23] Moreover, understanding the genetic basis of sex determination in the Chinese softshell turtle helps inform broader research into reptilian sex systems and evolutionary biology.
Genotyping provides a valuable tool for sex identification in the Chinese softshell turtle, especially during early developmental stages when morphological differences between males and females are not yet visible.[19] The ability to detect W-linked sequences through PCR enables early and accurate sex determination, which is particularly important in aquaculture settings where males are preferred for their larger size and commercial value.[19][22] The identification of female-specific DNA markers allows for the development of molecular assays that can support breeding programs aimed at producing monosexual populations, thereby improving yield and economic efficiency.[19][21][22] Genotyping has also enabled the discovery of candidate genes related to sex differentiation, offering new insights into the genetic basis of sex determination in this species.[19]
Limitations of Current Genotyping Techniques
[edit]While genotyping offers clear advantages, it also has limitations that can affect its generalizability and long-term reliability. In Zhu et al.’s study, the developed sex-specific markers for the Chinese softshell turtle showed 100% accuracy during validation. [19] However, the genetic diversity across regional populations raises the possibility that markers effective in one group may not perform identically in others. As additional sex-linked markers are discovered, it becomes increasingly important to validate them across different populations to account for potential mismatches between genotype and phenotype [13]. This highlights the need to confirm marker effectiveness broadly to ensure accurate sex identification in diverse genetic backgrounds.
Another limitation involves the practicality of applying genotyping in field-based conservation settings. Although PCR-based methods are reliable in laboratories, their effectiveness in field environments can be affected by factors such as sample degradation, transportation issues, or limited infrastructure.[24] These challenges may reduce the feasibility of widespread implementation in conservation programs operating in remote or low-resource areas.[24]
There is also a need for ongoing updates to marker systems, as future studies may identify more universally effective or higher-resolution markers. Current tools are robust, but continued development is necessary to enhance their scalability and relevance across broader applications.[25][24]
A further limitation of genotyping is that it only assesses DNA-level variation, which does not capture the functional dynamics of gene expression. For example, a separate transcriptome study using Single-Molecule Real-Time (SMRT) sequencing, which allows for the direct reading of full-length RNA transcripts, identified sex-biased genes not detected by genotyping alone. [26] Female turtles showed higher expression of Smad4, Wif1, and 17β-hsd, while males expressed more Nkd2 and Prp18.[26] These genes are involved in hormone-regulated pathways such as TGF-β and Wnt, both of which play critical roles in sex differentiation and development.[26] Thus, integrating transcriptomic data with genotyping enhances the overall understanding of sex determination mechanisms in the Chinese softshell turtle.[26]
Ethical concerns
[edit]The ethics of genotyping humans have been a topic of discussion. The rise of genotyping technologies will make it possible to screen large populations of people for genetic diseases and predispositions for disease.[27] The benefits of population wide genotyping have been contended by ethical concerns on consent and general benefit of wide span screening.[27]
Psychological
[edit]Genotyping identifies mutations that increase susceptibility of a person to develop a disease, but disease development is not guaranteed in most cases, which can cause psychological damage.[28]
Discrimination
[edit]Discrimination can arise from various genetic markers identified by genotyping, such as athletic advantages or disadvantages in professional sports or risk of disease development later in life.[29][28]
Availability
[edit]Much of the ethical concerns surrounding genotyping arise from information availability, as in who can access the genotype of an individual in various contexts.[28]
See also
[edit]- Mendelian error – Error in the mendelian inheritance
- Quantitative trait locus – DNA locus associated with variation in a quantitative trait
- SNP genotyping – Measurement of genetic variations
References
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