2. Materials and methods2.1. Fish u

2. Materials and methods
2.1. Fish used in the experiments
All the fish material used in this study originated from the National Breeding Center of Grass Carp, administered by Wujiang Aquatic Breeding Co. Ltd. (Jiangsu Province, China). The base populations examined in this work were previously collected from the Yangtze River in 2007–2008 (Fu et al., 2013b). These fish were physically tagged using passive integrated transponder (PIT) tags.
2.2. Mating and production of families
In May 2010, artificial fertilization was practiced separately in two batches (Batch 1 and Batch 2) with 52 or a 36 parents, respectively (as showed in Table 1). Each batch was implemented simultaneously with two breeding groups followed by mass spawning and partially factorial mating design (Gjedrem, 2005). Breeding groups GA and GB were implemented in the first (Batch 1), and GC and GD in the second (Batch 2) batch. For each group, the advance hormone-stimulated sexually mature males and females were maintained in a 20 m3 concrete spawning
tank. In the partially factorial mating groups (GA and GC), the eggs and milt were gently collected by hand stripping, and then eggs of a female were fertilized with a portion of milt from two different males. In the
mass spawning groups (GB and GD), newly fertilized eggs were collected through the outlet of the tank. For each breeding group, the fertilized eggs were pooled into iron incubators (ca. 1-tonne capacity) equipped with a
continuous flow-through of fresh water at 22–24 °C. After approximately two days, the hatched fry of each batch were communally reared separately in four nursery earth ponds (ca. 2000 m2 and 1 m depth) rich in
plankton. Fry stocking density was approximately 150-individuals/m2. At the fingerling stage (ca. 40 days old) and at first spring (ca. 10 months old), the stocking densities were similar to commercial rearing and adjusted to approximately 150 g total body mass/m2 initially, which contained approximately 75% silver carp (Hypophthalmichthys molitrix) juveniles polycultured together during these periods. At first spring (ca. 10 months old), the juveniles were randomly collected from each pond (1300, 1300, 800 and 1000 individuals, respectively) according to the amount of candidate parents for each breeding group (GA, GB, GC and
GD, respectively), and then mixed into one communal pond (ca. 4000 m2 and 1.5–2 m depth) for further rearing until artificial recording in the winter (ca. 18 months old). During the experiment, the fish fed commercial pellets (TongWei Company, China) to apparent satiation twice daily (at 8:00 am and 4:00 pm).
2.3. Data recording
In the first spring, a total of 960 individuals at the age of 10 months were collected randomly from the first breeding batch (Batch 1). In the second winter, a total of 2514 individuals at the age of 18 months were collected randomly from both two breeding batches (Batch 1 and Batch 2). Growth traits including body weight (BW), standard length (SL), body height (BH) and body thickness (BT) of fish were measured at the age of 10 months. Only two growth traits (BW and SL) of fish were measured at the age of 18 months, for saving time and lessen harm to fish. Meanwhile, fin samples from each fish were clipped and stored in absolute ethanol at 4 °C until DNA extraction.
2.4. DNA extraction
Genomic DNA was extracted using a traditional phenol-chloroform method. DNA concentration and purity were assessed using a NanoDrop 2000C (Thermo Scientific, USA) and by running a small amount on a 1%
agarose gel. DNA was diluted to 10 ng/μL, arrayed on 96-well PCR plates, and stored at −20 °C until further analysis.
2.5. Data analysis
2.5.1. Parentage assignment
Three multiplex PCR protocols based on twelve microsatellite loci were used for parentage assignment (Fu et al., 2013a) for all candidate parents and progenies. Pedigree reconstruction was performed with genotype data from all individuals pooled together in Cervus 3.0 software using the likelihood-based approach (Kalinowski et al., 2007).
The parameters for simulation analysis were as follows: 100% of candidate parents sampled, and 95% genotyped with a default typing error rate at 1%. In the parentage assignment analysis, less than three mismatched alleles were allowed for each offspring and a parent, and the unambiguously pedigree was generated in which only assignments with at least 95% confidence were accepted.
2.5.2. Preliminary analysis of growth trait values
Preliminary statistical analyses of data for growth traits were completed using SAS software (SAS-Institute, 1996). Data were assessed for normality (Kolmogorov–Smirnov test) and homogeneity of variances, and abnormal data were log-transformed (Ln) or square-root transformed before being used to calculate the variance components. Factors
(e.g. batch and group) were checked with the MIXED procedure using SAS software (SAS-Institute, 1996), and statistically significant factors were left in the mixed model for the estimation of variance components.
2.5.3. Genetic parameter estimates
The heritability (h2) and phenotypic and genetic correlations (rP/G) for growth traits were obtained from linear mixed models using the ASReml 3.0 software (Gilmour et al., 2009). For each trait, two different
animal models (Wilson et al., 2010) using restricted maximum likelihood (REML) algorithm were implemented for the genetic analysis as follows: