To edit the duck genome by HDR-directed integration of the EGFP gene into the duck host genome in combination with SMGT using CRISPR/Cas9. Methods. HDR-mediated gene of green fluorescent protein (EGFP) was crried out by the combined action of four plasmids. The pX330 contained the Cas9 gene. Two plasmids contained sgRNA genes: pBR322-sgRNA1 and pBR322-sgRNA2. The pBR322-HDR-EGFP plasmid was constructed to contain the DNA vector with left homologous sequence part(LHP), the EGFP gene coding sequences and the right homologous sequence part(RHP). The DNA sequence data for designing the HDR-EGFPinsert and sgRNA 1 and sgRNA 2 were taken from the genome DNA sequence of Anas platyrhynchos Spindlin 1 (SPIN1) gene. Twenty four ducks (13 males and 11 females) of the Shaoxing breed were used for this experiment. The sperm transfection was performed using Lipofectamine 2000. Results. Thirty one ducks were obtained, 19 of which carried the EGFP gene. F2 analysis revealed that 16 ducks (F1) (14 females and 2 males) transmitted the transgene DNA to their offsprings. Thus 27.6 % (56/203) of F2 descendants were positive for the transgene DNA construct. Conclusions. Exogenous DNA was successfully inserted into the duck genome.

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The purpose of this study was to monitor the egg productivity of the Shaoxing breed ducks from the age of 24 (beginning of egg laying) and 75 (end of egg laying) weeks. According to the results of a study of over 2385 eggs, a significant difference was found between the weight indicators (57.93 g and 70.81 g; p˂0.01), the shape index (73.71% and 75.35%, p˂0.01), the strength of the shell (4.92kg and 4.28kg; p˂0.01), the thickness of the shell of the egg (0.48mm and 0.45mm; p˂0.01) in ducks at the age of 24 and 75 weeks. It also shows changes in the weight and shape of the egg with the age of the bird. The necessity of further studying the genetic diversity of birds, which causes the separate variability of indices of individual ducks, is substantiated.

Microsatellite markers are now been widely used for the detection and description of micropopulation processes occurring in the populations of domestic animals for the effects of various factors of breeding pressure. Microsatellite loci distributed throughout eukaryotic genomes, making them the preferred genetic marker for high resolution genetic mapping. In recent years, rapid advances have been made in the development of molecular genetic maps. High-density linkage maps are now available for many farm animals, such as cattle, pigs, and goats. In contrast, mapping studies in avian species are much less advanced except in the chicken. According to FAO about 70% of ducks are bred in China. This country is a leader in growing ducks. The Shaoxing breed is one of the three major duck breeds in China. Ducks of this breed are characterized by high performance. According to the Bureau of Product Quality, the age of maturity (the beginning of egg laying) in these birds occurs at 130-140 days. The characteristics of the Shaoxing breed include the fact that the peak period of laying eggs lasts from eight to ten months. On average, one duck in 500 days gives from 290 to 310 eggs, which is one of the highest rates for egg breeds. That is why the purpose of our study was the microsatellite analysis of two populations of Shaoxing breed with 9 locuses was conducted. The selection of birds for the study were carried out on a duck farms in Zhejiang Generation Biological Science and Technology Co., Ltd. and Zhuji Guowei Poultry Development Co, Ltd., and at the laboratory of the Jjejiang Academy of Sciences Institute. Samples collection and DNA preparation: Venous blood samples were collected from 480 ducks (240 ducks of population I and 240 ducks of population II of the Shaoxing breeds) of both populations into 3 ml tubes containing EDTA as anticoagulant agent. In total of 9 investigated loci in the Shaoxing breed population, only one locus was monomorphic (SMO10). The number of different alleles (Na) for each polymorphic locus ranged from 2 (SMO12) to 13 (APL79, CMO11) in population I and from 2 (APL78, SMO12) to 7 (APL79) in population II. On average, one locus had 5.889 alleles in population I and 3.889 of alleles in the population II. The effective number of alleles (Nе) was 1.735 in population I and 1.599 in population II. The number of alleles and the expected heterozygosity (Hexp) values can provide important information for the discrimination of individuals and breeds. The index of expected heterozygosity in population I was 0.336 and 0.307 in population II. The information index (I) was 0,702 in population I and 0,576 in population II. For each population was found private alleles, in population I 6 alleles and in population II just 4 alleles. The results show high level of polymorphism of the studied populations of ducks. The obtained results can be used in the creation of new lines of ducks.

The consequences of chimerization and its possible influence on the productivity of chimera offspring remain poorly understood. The objects of research were ducks (Anas platyrhynchos) of the Shanma (Shan partridge duck) and Shaoxing breeds kept at the Zhuji Guowei Poultry Development Co, Ltd, P.R.China. The study was conducted in the poultry genetics laboratory of the Zhejiang Academy of Agricultural Sciences on a duck farm of Zhejiang Generation Biological Science and Technology Co., Ltd. (Zhejiang Province, PRC). To create chimeras of ducks, the method described by Aige-Gil, Simkiss, 1991; M.T. Tagirov, 2010 was used. Blastodiscs have been isolated from freshly hatched fertilized eggs using a filter paper ring. Shanma duck embryos have been used as recipients, and Shaoxing duck embryos, homozygous for plumage color gene allele (wild type), have been used as donors. Busulfan (SigmaAldrich, United States) have been used as a chemical agent that suppresses a division of primary germ cells (PGC) of recipient embryos. A hole in an eggshell (window) of recipients (Shanma breed) have been made between a blunt and sharp ends of eggs. (This reduced a distance between an injector and an embryo needle). The recipients havebeen incubated for 8–10 hours at a temperature of 38 °C. After recipient eggs incubation for 8 hours, the windows were opened in them. Busulfan was injected into the subgerminal cavity of the embryo with a micropipette (1.5–3 μl of liquid). After busulfan injection, the empty cavity was filled with culture medium (RPMI-1640) supplemented with antibiotics (ampicillin, streptomycin), the hole was closed by plastic wrap and adhesive tape. The eggs have been incubated at a reduced temperature (+32 °C) for 24 hours with the aim of prolong the duration of busulfan action on the PGC (primary germ cells). More than 50% of embryos have been died in the first 2–3 days (after an incubation start). Head and neck disorders have been observed in the 1.2% of embryos. Busulfan injection at a concentration of 300 ng per egg have been leads to 95.0–96.3% mortality of duck embryos, concentration of 150 ng per egg, a mortality rate of 33.3–75.3% have been observed, concentration to 75 ng led to 18.75–38.5% of embryonic mortality. Analysis of the age of puberty (laying of the first egg) indicates that the chimeras matured later. If in the control group the average age of puberty was 139 ± 9 days, in the group of chimeras - 148 ± 13 days. Thus, we can attest that in our experiment, the chimeras matured later than the control animals, which may be due to the effect of busulfan in the sterilization of recipient embryos. The average weight of ducks in the control group was lower, and the group itself was more consolidated. Thus, in the control ducks weighed 1422.40 ± 57.00 g, the chimeras 1608.80 ± 94.76 g. The advantage of live weight chimeras over the control group may be due to the fact that the control group consisted of recipients served by Shanma animals. Egg production of ducks for the entire study period was 87.5 ± 0.05 % (control) 79.5±0.12 % (busulfan). The weight of eggs of ducks of two groups for the entire period was 70.62±0.199 g (control) and 71.15±0.157 g (p˂0.001). The eggs morphometric parameters of the studied ducks groups were: the average values of egg length were 6.056±0.0564 cm (control) and 6.269±0.1341cm (busulfan); egg breadth were 4.520±0.0053 cm (control) and 4.529±0.004 cm (busulfan). There were no statistical intergroup differences in the morphometric parameters of the eggs of the studied groups. In fact, we obtained results similar to the previous ones, which concerned the egg production of daughters of drake chimeras.