GenotypinG-by-sequencinG (Gbs) as a tool for interspecies hybrid detection – a review

Genotyping-by-sequencing (Gbs) is an extremely useful, modern and relatively inexpensive approach to discovering high-quality single nucleotide polymorphisms (snps), which seem to be the most promising markers for identifying hybrid individuals between different species, especially those that can create backcrosses. In addition, GBS could become an invaluable tool in finding backcrosses, even sev - eral generations back. its potential for the use of restriction enzymes and species is almost unlimited. it can also be successfully applied to species for which a reference genome is not established. in this paper, we describe the Gbs technique, its main advantages and disadvantages, and the research carried out using this method concerning interspecies hybridisation and the identification of fertile hybrids. we also present future approaches that could be of interest in the context of the Gbs method.

Next-generation sequencing (NGS) is a DNA sequencing technology that has revolutionised genome research.Although it is less accurate than the standard Sanger sequencing method, it is much faster, less laborious and delivers huge data output (Behjati and Tarpey, 2013).What is more, in recent years, tremendous strides have been made in speed, read length, throughput and cost minimisation (Thermes, 2014), as well as computational data processing has become more routine, and computation tools are continuously improving (Kim et al., 2016).NGS platforms perform the sequencing of millions of small DNA fragments in parallel.Bioinformatics analyses link these fragments by mapping individual readings to reference genomes (Behjati and Tarpey, 2013).NGS sequence reads are a collection of a large, heterogeneous pool of DNA; for that reason, there is unavoidable variance in the sample sizes of individuals across loci and loci across individuals and uncertainty in genotype assignments between loci and individuals.However, the statistical packages for handling NGS data are constantly improved to keep these inconveniences minimal (Davey et al., 2011).
There are several genotyping methods that work based on NGS.They are aimed at developing and genotyping genome-wide genetic markers.The most popular of these use restriction enzyme (RE) digestion to reduce the complexity of the genome.These methods include sequencing using reduced representation libraries (RRLs), complexity reduction of polymorphic sequences (CRoPS), restriction-site-associated DNA sequencing (RAD-seq) and genotyping-by-sequencing (GBS) (Davey et al., 2011).
GBS is a simple, repeatable genotyping approach that allows a high level of sample multiplexing (De Donato et al., 2013).GBS makes it possible to sequence only a fraction of the genome while omitting repetitive genome elements (Narum et al., 2013).Unlike other methods with reduced genome representation, GBS has fewer steps and a simpler procedure (Elshire et al., 2011).Library preparation can be performed with small amounts of starting DNA, such as 100 ng and can be performed with high levels of multiplexing, making it possible to discover and genotype tens of thousands of single-nucleotide polymorphisms (SNPs) in one run and several individuals (Sonah et al., 2013).
SNPs are the most commonly used molecular markers due to their high density in the genome and the relative ease in determining their genotypes.SNP markers are often specific to the population for which they were developed.The traditional development of markers and their genotyping in populations has been a costly, laborious and time-consuming process, especially if a high amount of SNPs was analysed.Despite the advent of the SNP microarray and reduced workload, discovering SNP markers with the GBS technique is still competitive due to the lower analyses costs (Deschamps et al., 2012).Genotyping of one individual using a SNP chip with 250-760 K SNPs costs about 150-350 USD (Yue and Wang, 2017), while the GBS costs less than 30 USD per individual with multiplexing of 96 samples per lane (De Donato et al., 2013).GBS technique provides acceptable marker density at roughly one-third of the cost of currently available genotyping technologies.While SNPs arrays allow for the rapid collection of relatively inexpensive genome-wide marker data, they still require costly and time-consuming processes of SNP discovery, development, and implementation of the assay platform (De Donato et al., 2013).Using GBS, high coverage of SNPs in gene-rich regions of the genome can be easily achieved by selecting adequate restriction enzymes (Sonah et al., 2013).However, the usefulness of the data may be limited by various specific genome structures, such as repetitive DNA or variations in guanine-cytosine content, making the alignment of sequence reads difficult or impossible (Beissinger et al., 2013).Nevertheless, this method is a promising tool for the estimation of molecular markers because it reduces genome complexity and is capable of producing tens of thousands to hundreds of thousands of molecular markers (Poland and Rife, 2012).

Genotyping-by-sequencing
The first step in the GBS procedure is the restriction digestion of the genomic DNA of various individuals or populations treated as DNA pools.Following this, the resulting restriction fragments are selected and ligated to previously synthesized adapters, and then the samples are pooled and amplified.GBS uses barcodes of variable length (from 4 to 8 nucleotides), which are used for the computational separation of sequence reads belonging to different samples.PCR amplification ensures that the adapter-ligated fragments outweigh the other fragments.The fragments prepared this way are subjected to nextgeneration sequencing (NGS).The sequenced fragments allow the detection of polymorphisms, which serve as molecular markers.In this way, it is possible to discover and genotype informative SNP markers in one experiment (Davey et al., 2011;Elshire et al., 2011).
GBS makes it possible to analyse species with large genomes and those for which genomic information is lacking (Narum et al., 2013).However, when the reference genome is known, ordering and imputing low coverage marker data generated through GBS is easier.Nevertheless, genetic mapping developed with GBS can improve the reference genome.High-density genetic maps developed with GBS can anchor and organise physical maps and adjust or correct disordered sequence contigs.Reference genome development assisted by GBS markers leads to better SNP calling and development and refinement of a reference genome, creating a 'mutual loop' (Poland and Rife, 2012).Genotyping-bysequencing can be effective in species with high diversity and large genomes, reducing the complexity of the genome using restriction enzymes, which ensures sufficient sequence coverage.The use of methylation-sensitive REs makes it possible to avoid repeating regions of the genome and focus on target regions with a lower copy using two-to three-fold higher efficiency (Elshire et al., 2011).GBS applies RE digestion to preferentially target sites in low-copy-number regions of the genome, minimising reads in repetitive sequences (Beissinger et al., 2013).
For species with large genomes or without a reference genome, rarely cutting methylation-sensitive restriction enzymes help reduce genome complexity by acting on fewer sites.In the absence of a reference genome, it is required to identify a pair of nearly identical readings (Torkamaneh et al., 2016), while the reference map can only be developed near the restriction sites, which can be done in the sample genotyping process, and the readings obtained become the reference point.Moreover, the sequence tags can be considered dominant markers (Elshire et al., 2011).Therefore, GBS does not require knowledge of the genome of the analysed species since SNP discovery and genotyping occur simultaneously (Kim et al., 2016).The unusually large number of markers obtained from GBS allows sufficient coverage of the majority population (Poland and Rife, 2012).
Compared to other methods, reducing the complexity of the genome by GBS is easy, fast, specific, reproducible and makes it possible to reach regions that are difficult to capture (Elshire et al., 2011).Moreover, the original GBS protocol used a single restriction enzyme, but the combinations of enzymes that can be used to reduce the complexity of the genome are almost endless (Poland and Rife, 2012).
GBS is a modern molecular approach with many advantages.The main goal of GBS is to discover polymorphisms and obtain genotypic information in the entire study population, which makes it a quick and flexible approach (Poland and Rife, 2012).It makes it possible to obtain highly informative SNPs at a lower cost than using ready-made microarrays (Beissinger et al., 2013).GBS libraries need a very low coverage of NGS reads; thus, the cost of sequencing is relatively low compared to other methods.A much simpler experimentation procedure itself makes it not only easier to generate restriction fragments with the right adapters, but the digestion of genomic DNA and ligation of adapters occur in one well, there are fewer DNA purification steps, and fragments are not size selected (Elshire et al., 2011).Thus, the steps needed to prepare a library for sequencing are minimal (Gurgul et al., 2019 a).In addition, GBS can be very effective in mapping single genes.This means it is possible to identify markers related to the function of a particular gene (Poland and Rife, 2012).Additionally, raw sequences obtained from GBS can be reanalysed, revealing further information as bioinformatics techniques improve, reference genomes develop and the amount of sequence data increases (Poland and Rife, 2012).
Despite the benefits of GBS, it is not without drawbacks that are important to consider in the experimental design stage.There is a risk of errors in sample preparation and sequencing in the GBS routine (as well as in all NGS techniques), leading to variable sequencing coverage (Deschamps et al., 2012).One disadvantage of GBS is that datasets often contain many missing data points due to low-coverage sequencing; therefore, in the case of the GBS, generating more sequence reads provides more data per sample and is preferable to generating longer reads (Poland and Rife, 2012).It also happens that not all sequenced regions are evenly covered for all individuals in a population (Deschamps et al., 2012).Moreover, the depth of coverage is important for assessing polymorphism (Ellegren, 2014).In addition, traditional Sanger sequencing or SNP genotyping methods are more accurate than data obtained from NGS, on which the GBS is based.Searching for SNPs in a population using this technology is also problematic since NGS produces shorter reads than Sanger sequencing, increasing the risk of matching a specific sequence to the wrong region of the genome.Nevertheless, with the advancement of NGS techniques, algorithms have been introduced that deal with problematic data and correct the resulting errors (Deschamps et al., 2012).Moreover, reads depth per locus can be enhanced by reducing the number of genotypes per library, using rare-cutting restriction enzymes, double digestion and multiple library sequencing (Kim et al., 2016).

interspecies hybridisation
Hybridisation is reproduction occurring between genetically distinct populations and is inextricably linked to the production of offspring of mixed origin.It can cause interactions spanning a wide range of genetic divergence between parental forms (Abbott et al., 2013).Hybridisation can occur spontaneously, but in most cases, it is anthropogenic (Yadav et al., 2019).Recently, it has been proven that hybridisation and gene flow between species is much more widespread than previously thought (Pfennig, 2021).It is estimated that hybridisation affects as many as 10% of animals (Adavoudi and Pilot, 2022).The causes of hybridisation are usually a sudden reduction of the population, the reduced availability of mating partners, the destruction of species' natural habitats, the introduction of a new species to a given habitat, the migration of species or even deliberate human activity (Yadav et al., 2019).This phenomenon can indirectly increase genetic variation by increasing the mutation rate (Marques et al., 2019).
The resulting interspecies hybrids are usually infertile and sometimes even inviable due to the varying number of parent chromosomes.A different number of chromosomes makes it impossible to produce efficient gametes.In the case of hybrid individuals, males are often more sterile than females (Yadav et al., 2019).Nature has developed various safeguards to limit interspecies hybridisation in the form of different mating seasons, differences in sperm acrosomes and in the intrauterine environment, and even the enzymes required by sperm to penetrate the egg cells, treating the sperm of a species as a foreign body by the female of another species (Gabryś et al., 2021;Yadav et al., 2019).
In some cases, hybrids are fertile, and this causes negative effects for the most part.F 1 hybrids can produce F 2 offspring by mating with other F 1 hybrids or backcrossing with the parent species (Allen et al., 2020).In some cases, hybrids are free to cross with both parent species.
It happens in some closely related animals having the same number of chromosomes.Such events lead to introgression (Yadav et al., 2019).
Human activities through urbanisation, habitat destruction, predator control and poaching directly contribute to interspecies hybridisation (Germain et al., 2008).In some cases, interspecific hybridisation can increase extinction risk for threatened species (Forsdick et al., 2021).As backcrossing progresses, the morphological differences between the parent species and the backcrosses decrease, making it increasingly difficult to detect backcrossing using only phenotypic traits (McFarlane and Pemberton, 2019).

application of Gbs in interspecies hybrid detection
Research on interspecific hybridisation and introgression continues to be a topical and controversial issue affecting the conservation and genetic diversity of different species (Germain et al., 2008).The improved resolution provided by genome-wide markers leads to increased backcrossing and introgression detection.The use of high-density markers to study genomic heterogeneity allows for greater genetic discrimination between closely related hybridising species (McFarlane and Pemberton, 2019).Research identifying specific hybrid individuals seems to be required due to the improvement of protection programs and the preservation of the genetic diversity of hybridisable species (Germain et al., 2008).Therefore, genome-wide methods such as GBS seem to be a promising solution in this case.It seems that it may be a potential tool to detect specific hybrid individuals by estimating specific population genetic relationships, significantly increasing the number of markers tested.

Birds
Studies on the interspecific hybridisation of birds have been performed on two related species, the Syrian woodpecker (Dendrocopos syriacus) (SW) and great spotted woodpecker (D. major) (GW) (Gurgul et al., 2019 b).In this case, it was impossible to map reads to the reference genome because such a genome was not available.In this study, a panel of 2479 informative SNPs was generated, and it was found that hybridisation between SW and GW occurs much more often than estimated in previous genetic studies involving microsatellite and mitochondrial DNA markers.The results have shown that some SW individuals have features of the GW genome, which suggests hybridisation and introgression.Despite the lack of a reference genome, GBS produced an adequate number of polymorphisms and contributed to a significant elimination of costs associated with genotyping microarray assay, which would be unprofitable in wild birds.The authors suggested that hybridisation and backcrossing with GW resulted in a high reduction of the characteristic SW alleles in the genomes of birds from urban populations.
Three species of woodpeckers in Canada intersect with each other: Sphyrapicus varius, S. nuchalis and S. ruber (Seneviratne et al., 2016).These three species differ in expressive patterns in red, white and black.The conducted analyses made it possible to conclude that the phenotypically divergent S. ruber and S. nuchalis are closely related genetically, while S. nuchalis and S. varius have similar plumage but are more differentiated.Within each zone of mutual occurrence of these species, there was clear evidence of hybridisation, but both plumage data and genotypic data indicated that the hybrid frequency in each zone was relatively low.It has been suggested that this indicates some form of decreased hybrid adaptation or a strong negative selection acting on the resulting hybrids.
Another example of the effective use of the GBS method was the assessment of hybridisation, especially the degree of introgression to date between the species of two closely related birds -one of which is critically endangered with extinction (Forsdick et al., 2021).The black stilt (BS) (Himantopus novaezelandiae) is one of the rarest birds on earth and, unfortunately, capable of hybridising with the fairly common species of the white-headed stilt (WHS) (H.himantopus leucocephalus).The analysis proved that, despite the hybridisation, there was no introgression in the BS genome, which confirms previous studies using microsatellite markers.Moreover, it has been suggested that even a few generations of backcrossing with BS could quickly overwhelm the WHS introgression.In this case, hybridisation could lead to genetic flooding and, thus, the extinction of the endangered species.It has been suggested that with the arrival of the WHS to the BS sites, BS genetic material was hybridised and introgressed, which was maintained in later generations despite backcrossing due to the initially small number of WHS specimens.

Reptiles
In a GBS study on rattlesnakes, it was possible to generate informative SNPs that made it possible to prove hybridisation and introgression between several species of rattlesnakes, with a relatively small number of samples taken (i.e., 58) (Myers, 2021).The Western rattlesnake species group (Crotalus viridis species complex, C. mitchellii species complex, C. scutulatus and C. tigris) was tested.It has been suggested that all species have hybridised to at least one other lineage, and these events are mainly confined to species that coexist geographically.One notable exception was identified by the detection of gene flow between C. oreganus and C. tigris, whose distribution is >185 km apart, suggesting hybridisation events in the distant past.These phenomena hinder the phylogenetic analysis and diversification assessment among these species.Introgression and gene flow in this group make it increasingly difficult to distinguish individuals from a particular species.Phenotypic variation within species, combined with extensive gene flow between genetic lines, is likely to confuse species identification and diagnosis.Moreover, the strength of the introgression signal decreased with geographic distance but was always detected.
The leaf-toed geckos' species (Phyllodactylus) found in Mexico likely contains more than 30 species (probably 35) (Ramírez-Reyes et al., 2020).The high level of GBS resolution was sufficient to demonstrate an underestimation in the number of species based on morphological data alone.Moreover, the authors reconstructed the evolutionary history of Mexican leaf-toed geckos, estimated the phylogenetic relationships, and found evidence of a genetic admixture between several species.This species included: P. bordai and P. papenfussi; P. lupitae with individuals from the Nayarit coast; some P. nocticolus with P. x. angelensis; P. t. saxatilis with populations from Mocuzari and Maria Cleofas; P. t. saxatilis with P. partidus and P. homolepidurus.These observations led the authors to hypothesize that genetic exchange occurs between the insular and mainland populations of leaf-toed geckos.
The genomic admixture between the American Crocodylus acutus and the Morelet's species C. moreletii was also investigated using GBS (Pacheco-Sierra et al., 2018).It has been suggested that hybridisation has been going on since ancient times.Additionally, individuals collected as C. acutus were, in fact determined to be two separate species.The authors suggested that a taxonomic reassessment be performed.Moreover, it has also been shown that most of the populations considered to be C. moreletii are hybrids.

Fishes
Hybridisation also takes place between species of zander (Sander vitreus) and sauger (S. canadensis), creating fertile 'saugeye' hybrids (Graham et al., 2021).The crossing of the two species is much more frequent in artificial reservoirs.However, spontaneous hybrids have also been observed in nature.In these two species, the crossing of F 1 hybrids with one another is rare, while the crossing of F 1 hybrids with one of the parent species is much more frequent, which unfortunately contributes to introgression.Graham et al. (2021) showed that the parent species were classified with high accuracy.Researchers have shown that in both main reservoirs of the Saskatchewan River in Canada, spontaneous hybridisation of zander and sauger occurs.GBS enabled the identification of many generations of backcrosses with both zander and sauger and found evidence of an F 2 hybrid (F 1 × F 1 cross), thus proving that they are viable because this was only the second such specimen recorded in nature.The authors also suggested that introgression may prevent the phenotypic differentiation of hybrids from the parent species, ultimately leading to a disorder of genetic diversity.These studies turned out to be important for breeding reasons, as they indicated that breeders should not collect breeding material from the examined sites in order to stock other reservoirs.
Similarly, it was possible to prove for the first time hybridisation and introgression between two closely related species of sharks: Galapagos (Carcharhinus galapagensis) and dusky (C.obscurus) (Pazmiño et al., 2019).Both species show morphological similarities.Using GBS, the authors obtained 1,873 highly informative SNPs capable of detecting low levels of admixture, allowing for the separation of these two species and the finding of common hybrids, which showed high discrimination power at the level of FST = 0.47.Moreover, they proved that these species could not be differentiated with the use of mtDNA, suggesting that hybridisation had occurred among distant ancestors of the present population.However, using GBS, it was possible to identify four specific hybrids and demonstrate bidirectional introgression, which constituted about 1% of the total sampled population.Further, it was proved that hybridisation does not occur in places where these species are common, and it is not difficult for individuals to find a reproductive partner within their own species.Because shark populations are decreasing worldwide, the authors suggested that hybridisation phenomena will become increasingly common in the future.
Cases of hybridisation between two species with different mating systems were found in Kryptolebias ocellatus and K. hermaphroditus in two hybrid zones in Southeast Brazil (Berbel-Filho et al., 2021).The time of self-pollination and unilateral incompatibilities, in this case, was found to play a big role.K. hermaphroditus, mangrove killifish, is one of two known examples of the self-fertilisation of vertebrate hermaphroditism.It has been suggested that K. ocellatus is an exclusively outcrossing species.With the use of GBS, all F 1 hybrids from these two species were proven to be descended from male self-pollinating K. hermaphroditus species.The hybrid zone between K. ocellatus and K. hermaphroditus is representative of asymmetric or unidirectional hybridisation, and evidence also indicates asymmetric backcrossing.It has been suggested that an unusual hybridisation phenomenon is the result of water pollution, which interferes with physiology, mate selection and reproductive isolation between coexisting species.The waters in which these species live are among the most polluted in Brazil.

Insects
In the course of studies on phylogenetic relationships between various species of Choristoneura sp.(Dupuis et al., 2017), evidence of hybridisation and introgression between members of the spruce budworm group was observed, and the species C. lambertiana was described as an intermediate form.Sampling in this experiment was designed to capture as much geographic variability as possible, and 66,349 SNPs were obtained using the GBS approach.The researchers proved that C. fumiferana, C. pinus and C. retiniana formed monophyletic groups, while C. oc.occidentalis, C. oc.biennis, C. carnana, C. lambertiana and C. orae showed clustering ambiguity, suggesting hybridisation events.
Following interesting example was the analysis of the hybridisation of butterflies of the Papilio machaon species group (Lepidoptera: Papilionidae), in which six or seven species are recognised, and hybrids mean that the phylogenetics of these species is still not well understood, and the hybridisation contrasts with the detailed analysis in this direction (Dupuis and Sperling, 2020).Previous studies have already shown the existence of hybrids among these species, but SNP markers obtained with GBS have allowed for new hypotheses.It has been suggested that hybridisation phenomena occurred between the two species in this group already during the transitional Pleistocene glaciations due to unexpected contacts of these species; namely, a paraphilia was found between P. machaon and P. zelicaon.The presence of hybrids between P. hospiton and P. machaon, which previous studies had not reported, was also found, while estimating that the current gene flow is relatively low.The study also confirmed the earlier information, that is, the existence of 3 hybrid lineages: P. brevicauda, P. joanae and P. m. kahli.A close relationship was demonstrated between these hybrid lineages and the hybrids of P. zelicaon or P. machaon × zelicaon.The studies carried out suggested that some nuclear signatures within these hybrid genetic lines link them to the original maternal species as well as their original paternal species.
Another example is the hybridisation of Helicoverpa armigera with H. zea, which may result in losses in agricultural production (Cordeiro et al., 2020).The former feeds mainly on soybeans and cotton, while the latter feeds on corn.The invasive species H. armigera is characterised by a rapid evolution of resistance to insects, and therefore it is called the 'world mega pest'.It was found that hybridisation varied significantly in the degree of introgression depending on the sample location, landscape composition and climatic conditions.It was also found that the hybrid zones are mainly related to the cultivation of maize and soybeans, with the level of hybrids ranging from 15 to 30%, depending on the sampling location.Crop rotation has also been found to be particularly problematic and to increase the likelihood of crossbreeding; hybridisation itself may result in increased caterpillar feeding pressure on soybean and introgression of pesticide-resistance genes.
future perspectives GBS has proven many times that it is a good approach to detect hybrids and identifying introgression.The problem of hybridisation among animals is so great that it would even be advisable to use GBS in research on hybridisation and the level of admixtures in individuals in some species.One of the significant problems is the hybridisation of South American Camelids (SACs), which include two wild species: guanaco (Lama guanicoe) and vicuña (Vicugna vicugna), and two domesticated species: llama (L.glama) and alpaca (V.pacos).All SACs have the same number of chromosomes (2n = 74) and can cross, producing fertile hybrids (Podbielska et al., 2021;Wheeler, 1995), and this phenomenon occurs especially often among domesticated species (Wheeler et al., 1995).The first generation of alpaca × llama hybrids is easy to recognise, but the most problematic is backcrossing (Di Rocco et al., 2011).It is an unfavourable phenomenon that threatens genetic diversity and causes a significant deterioration in the quality of alpaca fibre, which is considered a luxury material (Podbielska et al., 2021;Wheeler et al., 1995).It has been proven that, despite various breeding practices, alpacas and llamas have not yet divided genetically, proving the phenomenon of hybridisation identified based on STR markers (Echalar et al., 2020;Echalar and Barreta, 2022;Podbielska et al., 2021;Varas et al., 2020), mtDNA (Barreta et al., 2013;Kadwell et al., 2001;Maté et al., 2007Maté et al., , 2004;;Melo et al., 2012;Podbielska and Piórkowska, 2022;Stanley et al., 1994) or even the Y chromosome (Marín et al., 2017).Probably also important in this case is the fact that the hybridisation of alpacas with llamas influenced the genomes of both species (Wheeler, 2012).In these two species, it seems justified to use SNP markers to identify hybrids.GBS could also be an ideal solution because an improved alpaca reference genome is available (Richardson et al., 2019).Multigenerational hybrids are the main problem among alpaca and llama species.Recent studies involving the SACs genome (Fan et al., 2020) have been aimed at determining their origin and ancestry, but to our knowledge, no genomic studies (e.g., GBS) have been performed to identify specific hybrid individuals of these species.To date, a bovine array has been tested to identify SNP markers in alpacas (More et al., 2019), and a 76k microarray was designed that is specifically dedicated to alpacas (Calderon et al., 2021).However, the authors did not test these tools for hybridisation detection.Nevertheless, the cost of using microarrays exceeds the cost of independent GBS analysis and will find more SNP markers randomly placed in the genome (Kim et al., 2016;Yue and Wang, 2017).
Because alpacas were classified as livestock in Poland in 2021, a good breeding approach would be to systematise breeding and trying to search for hybrid individuals, especially those difficult-to-identify backcrosses.Their elimination from reproduction could effectively affect the fibre quality of future populations (Podbielska et al., 2021).
Therefore, it is assumed that in the detection of multigenerational crosses of alpacas and llamas kept in Poland, the GBS technique and the generation of highly informative SNP markers that would indicate specific hybrid individuals would be effective.It was not possible when examining the mtDNA and Y chromosome (Podbielska and Piórkowska, 2022).When using STR markers, the F 1 hybrid had only 7.4% llama admixture, suggesting that alpaca and llama populations are genetically mixed (Podbielska et al., 2021); however, it is hypothesised that the SNP markers may be much more precise.conclusions GBS was developed and applied in the sequencing of multiplexed samples, and it combines molecular marker discovery and genotyping using NGS.In the last few years, it has been proven that GBS can be successfully used not only for plant genotyping -for which it was originally developed -but also for animal genome genotyping.It has also been shown that it is possible to use GBS to generate informative SNPs throughout the genome, making it an excellent tool for the assessment of the degree of hybridisation, particularly given that it allows for the identification of backcrosses.In addition, its potential for the analysis of various species is almost unlimited, as it can be successfully used even in species without a reference genome.