The genomics community has been enormously revolutionized by first- and next generation sequencing (NGS) technologies [1–3] that generated vast amounts of genetic data ranging from unicellular (bacteria) to multicellular (eukaryotic) genomes. However, no matter how good these technologies are, polymorphisms in a genome complicate the assembly process, results in lower quality, and the contiguity and completeness of assembly is significantly lower than would be expected from a homozygous template . Hence, there is a growing interest for the development of inbred lines, such as haploid and double haploid (DH) lines, which are particularly advantageous in genomics [5, 6] because of their homozygosity and their growth potential. Inbred lines are also useful in physical mapping  and in genetic mapping  which would enable precise positioning of the genes in the genome, and thus facilitate accurate identification of candidate genes. Furthermore, DHs are also useful for mutagenesis and genetic transformation studies [9–14].
An inbred line is normally produced by classical sibling mating which is not only time and space consuming, but also laborious and expensive, especially in organisms with long generation times. Alternatively, it can be produced via androgenesis or gynogenesis which both requires inactivation of the genetic material through chemical means or by ionizing radiations. For species with relatively large eggs (1.5 - 1.7 mm), with thick membrane that hardens upon contact with water and contains numerous oil globules like that of the three-spined stickleback, optimum inactivation of the genetic material of the eggs is difficult and hence gynogenesis should be favored. Gynogenesis is defined by Thorgaard  as an all maternal type of inheritance wherein the genetic material of the sperm does not contribute to the resulting embryo. This process occurs naturally in some fish species like in crucian carp and in several species of poecilids [16–18]. Induction of diploid gynogenesis involves egg activation by irradiated homologous or heterologous sperm, and diploidization by retention of the second polar body (meiotic gynogenesis) or suppression of the first mitotic cleavage (mitotic gynogenesis)  using strong physical treatments (i.e., shocks). The most commonly used treatments are either low or high temperatures (cold or heat shocks) or hydrostatic pressure. Under the influence of such treatments, the spindle fibers are destroyed and cell division stops resulting in the fusion of the daughter cells, thus forming diploid gynogenetic embryos. Mitotic gynogenesis was regarded as the fastest way to produce DH [completely inbred (homozygous) fish]. It requires only one round of gynogenetic manipulation. Production of DHs via meiotic gynogenesis, on the other hand, requires an additional 2-3 rounds of gynogenetic manipulations. Prior to the boom of the genomics era, artificial gynogenesis has already been applied in many fish species mainly because of its potential value in experimental genetics and aquaculture [20, 21]. Furthermore, it has played an important role in fish genetic improvement and control of reproduction . For instance, diploid gynogenetic fish were observed to grow faster and have stronger resistance to disease than haploids . Successful gynogenetic manipulations were done, e.g., in rainbow trout , medaka , common carp , muskellunge [25, 26], goldfish , Russian sturgeon and starlet , mud loach , sea lamprey , and in Wels- and channel catfish [6, 31].
The three-spined stickleback is a teleost that exhibits multiple examples of parallel evolution. It inhabits different marine, estuarine, and freshwater systems all over the Northern hemisphere and is characterized by rapid and repeated ecological and phenotypic divergence from the marine ancestor to the freshwater forms [32, 33]. Like other model systems, the linkage and chromosome maps of this fish have already been published [34, 35] and its genome sequenced . Genomic information has now been used to understand many fundamental evolutionary problems. For instance, the successful elucidation of the evolution of body armor reduction in three-spined sticklebacks from the phenotypic down to the gene level  prompted Gibson  to elevate the status of this species to an evolutionary supermodel. The advent of NGS has also advanced studies on the population genomics of this species . However despite all these developments, the problem with polymorphism remains and left a significant part of the genome unassembled. Thus, increasing the need for the production of an inbred three-spined stickleback.
In our knowledge, no inbred line is available in this species partly because the emphasis has always been on the study of natural populations. Information about effective modification of genotype in this species was limited only to ploidy manipulation using temperature shock that resulted in the production of triploid and haploid individuals [40, 41]. These haploids would be ideal for genomics but as in many attempts of fish haploid production, these haploids survived only until embryonic development [20, 42–46] or right after hatching [10, 47, 48] and hence do not provide enough template material for downstream processing.
Here, we present a working protocol for the induction of gynogenesis using a combination of sperm UV irradiation and heat shock (HS) treatment. This study aims at producing inbred lines of the three-spined stickleback suited not only for whole genome sequencing, physical and genetic mapping but also for future mutagenesis and experimental studies. Success of gynogenesis is confirmed by microsatellite DNA analysis and the ploidy of the gynogenetic embryos/larvae is verified by flow cytometry.