This study describes the methods and production of the first cloned Atlantic salmon lines. In two experiments fertilization with UV irradiated sperm combined with a pressure shock applied at 4700 - 4800 minC at 8 oC gave all homozygous (doubled haploid) gynogenetic Atlantic salmon, with survival rates ranging from 9.6 to 16.7% after 107 to 180 days of feeding. Based on the absence of paternal inheritance found in the microsatellite loci and the female sex of the progeny (Supplementary file 1), we conclude that both UV protocols (6 and 8 mins) used in Exp2011 gave a complete inactivation of the sperm DNA. Also, the homozygosity for all investigated loci in the progeny show that they are produced by first cleavage block gynogenesis and not spontaneous polar body retention. A blockage of the second meiotic division would leave some level of heterozygosity , as the female progenitor had a high level of allelic heterozygosity (15 out of 18 of the investigated loci).
The timing of the pressure shock in Exp2011 (3800 to 4800) were chosen after  who produced mitotic gynogenetics when activating salmon eggs with UV irradiated sperm and using pressure shock at 400 to 470 mins after fertilization at 10 oC, finding an optimized timing at 440 mins (4400 minC). In our two experiments we found an optimized induction time around 4700 to 4800 minC, corresponding to between 75.8 and 77.4% of the FCI (using the 6200 FCI found in Exp2011 which agrees well with the FCI of 6240 described earlier .
Optimal timing of a temperature or pressure shock has earlier been shown to coincide with the metaphase [2, 5, 48, 49] and/or prometaphase  of the mitosis, preventing the partitioning of the duplicated chromosomes into two cells. The result is a cell with two identical sets of chromosomes : doubled haploids when using UV irradiated sperm, or tetraploids when using intact sperm. Our optimized timing agrees well with studies done on several other species, reported to be 65±5% of the FCI in rainbow trout , 70-72.5% in brook trout , 70-75% in common carp , but differs from 45.8-57.2 % in pressure-shocked Nile tilapia [53,54]. However, variation between studies and species exist as FCI is dependent on temperature , increases during the spawning season and with postovulatory ageing of the eggs , can vary between populations [55, 56], from year to year in the same population  and varies between females .
The survival until first feeding of doubled haploids was considerably higher in Exp2011 (22-24% of controls) than in Exp2012 (12.3% of controls), however, both were in the range of the average of 19% found in rainbow trout  and 14-16% surviving swim-up fry in brown trout . In studies on other species, survival varies considerably. Twentynine % normal-appearing embryos were found 24 hours after fertilization in zebrafish , compared with 0-6% survival until hatching in Medaka , 3.5-15% normal fry 96 hours after fertilization in common carp , <1-26% feeding fry varying between females in loach (Misgurnus anguillicaudatus) , 6.9% of control at yolk sac resorption in Nile tilapia  and 12.8% until hatching in red sea bream . However, these studies do not represent long-time survival as doubled haploid progeny often suffer high mortalities during early life. In  the progeny of the six best females of 15 had a mean survival until first feeding of approximately 30% which was reduced by approximately one third to 20 % during the first 150 days of feeding, comparing well with the mortality of approximately 30% during the first 107 days of feeding in the present study (Table 1). Still this survival is high compared to other species, with 5.8% survival until adults in zebrafish , 0.2% in medaka , mortalities between 36.1 and 57.3% between hatching and 28 days post-hatching in common carp , mortality of 91.4% at 90 days in red sea bream  and survival of 155 of 323 hatching doubled haploid progeny in Nile tilapia . The survival of doubled haploid progeny from 15 rainbow trout females varied between 0 and 53%, and it was hypothesized that genetic factors could explain part of the variation . Generally, the main factors influencing on the yield/survival of doubled haploids are species-specific gene dosage compensation mechanisms, expression of early embryonic recessive homozygous deleterious mutations, egg quality and the occurrence of spontaneous absorption of the polar body creating heterozygous meiotic diploids [see reviews 3, 9]. In the present study the egg quality was good (high survival of controls) and no heterozygous meiotic diploids were observed, giving support to the hypothesis that genetic factors, i.e. occurrence of recessive deleterious alleles, was the main reason for the reduction in early survival.
In the present study, no significant differences in mean weight were found at any time between the control group and the treatment groups or among the treatment groups. However, our groups are based on one female progenitor. In rainbow trout, the mean weight of groups of mitotic gynogenetics were less than 80% of that of the diploid controls after 103 days of feeding , with the reduction varying between 4.1 and 30.9% dependent on the female progenitor. Also, the much higher variation than in the controls, the high frequency of small fish, the significantly higher condition factor, and the higher incidence of individuals with curved or shortened vertebral column in the doubled haploids agrees well with earlier studies. Red sea bream mitotic gynogenetics were compared with diploid controls for almost three years following hatching and the gynogentics had lower weight from year one on, a higher body depth, a much higher variance in measured parameters, and a higher incidence of short vertebral columns, scoliosis and deformities in the head . In carp mitotic gynogenetics had lower weight and higher variance compared to controls  and doubled haploid tilapia had high incidence of deformities and retarded growth . Our radiological examination in June 2013 revealed that 4 out of the 30 radiographed dh individuals had vertebral deformities which can affect the condition factor [see 64]. However, the number of affected fish and the low number of affected vertebrae is too low to explain the observed differences in condition factor between the doubled haploids and controls. The low incidence of deformities and also the fact that some of the doubled haploid individuals are heavier than the controls in November 2012 (Figure 1B), indicate a potential for production of well-performing doubled haploids with a normal phenotype.
Production of clonal lines
In this study five all homozygous clonal lines were produced by meiotic gynogenesis of eggs from doubled haploid progenitors and verified as clonal and identical to their mother with microsatellite markers.
Poor reproductive performance of doubled haploid progenitors has been highlighted as a major limitation in the production of isogenic lines [9, 60]. The relative fecundity (eggs/kg female bodyweight) of doubled haploid rainbow trout was normal and even significantly higher than controls , but with observations of sterile and hypofertile fish, and homozygous carp females had severe gonadal defects and less than 10% could be reproduced . In our study, egg production of the female progenitors was high and well within what is normally seen in outbred Atlantic salmon [e.g. 65, 66]. However, early mortality was high, in accordance with studies on other species; e.g. between 3.3 and 36.9% of controls in tilapia , less than 5% survival until yolk sac resorption in Nile tilapia , between 0–70% survival until hatch in medaka , and 29 and 50% survival until first feeding at first and second spawning in rainbow trout .
The treatment protocol that is used, i.e. the UV-irradiation protocol of the sperm which can leave fragments of chromosomes [58, 69] and the pressure treatment of the eggs  can potentially contribute to the mortality in the clonal lines as well as in the production of the dh progenitors. However, both the UV-irradiation protocol and the pressure treatment protocol that was used in the present study are strictly standardized and cannot explain the variation in survival between the lines. Moreover, the pressure treatment protocol is used extensively in production of triploids both for aquaculture production and for research purposes. Different triploid induction protocols have been tested in brown trout  and Arctic charr (Salvelinus alpinus) , and despite a high variation in the parameters (pressure, timing and duration of pressure) they found a high triploidisation rate, and a survival that is high, and only occasionally significantly different from controls. If the pressure shock itself had some detrimental effect on egg/larval development and survival, one would expect this to be consistent over studies and in the case of our study give the same effect in all lines. Neither should the early mortality in production of homozygous clonal lines be influenced by lethal alleles because they have been eliminated in the first generation . However, each line still represents only one haplotype (extreme inbreeding) and can contain homozygous alleles that are detrimental which again can be mirrored in the variation in phenotype and survival within and between lines as seen in the present material. Also, in our study all the dh females were stripped for eggs on the same day and the time of ovulation was not recorded. Hence, as post-ovulatory aging is an important determinant for egg quality (e.g. [72, 73] this could be an important variable leading to differences in survival. The importance of egg quality has also been demonstrated in rainbow trout where survival until first feeding increased from 29% in first time spawning doubled haploids to 50% in their second spawning . Also, both the extreme inbreeding and possibly also a post-ovulatory aging of the eggs are factors that can contribute to the different morphological deviations and deformities that were seen in our clonal lines. The effect of inbreeding on body deformities is well described in studies on both salmon  and rainbow trout  and in fully homozygous fish the incidence can be considerable [see 69]. However, in salmon, deformations linked to post-ovulatory aging were mainly found in the head  and these were not seen in the present study.