Israeli carp (C. carpio) fishes were collected from Chungnam (KorA, 47 samples, 2–3-year-old) and Jeonbuk (KorB, 51 samples, 2–3-year-old) in Korea, and samples of cultured Chinese Israeli carp (C. carpio) were collected from Jilin province (ChA, 76 samples, 2–3-year-old) and Heilongjiang province (ChB, 80 samples, 2–3-year-old) in China (Table 1).
Basic morphological analysis
A total of 98 of Korean Israeli carps and a total of 156 Chinese Israeli carps were measured using a ruler and Vernier calipers for total length, body length, weight and body height. Condition factor (CF) and body shape were calculated using the following formula: CF; Body weight(g)/Total length(mm)3 ×. Fish were classified into 5 classes based on a 5-step scoring system of scale distribution: 1 score (0~3 scales), 2 score (4~10 scales at some dorsal fin scales), 3 score (14~17 scales at dorsal fin and tail), 4 score (18~33 scales at dorsal fin, tail and sideline) and 5 score (≥34 scales at dorsal fin, tail and sideline and whole abdomen). RFID (Radio Frequency Identification) tags were inserted into all individuals of the broodstock, and the morphometric analysis each individual was managed by measuring the total length, body length, weight, CF, body shape and scale value according to each unique number. The collected broodstocks were anesthetized for genetic analysis and morphological measurement. The groups were anesthetized by immersion in 100mg/L MS-222 (Sigma, MO, USA) diluted in water. After the fin (1cm2) was taken and stored in ethanol tube (99.9%). RFID tags were inserted into all individuals of the broodstock. Immediately after the measurement, they recovered from anesthesia in the recovery tank. And then, they were sent to the rearing tank for management.
Broodstock selection and mating scheme
The breeding experiments of cultivated Israeli carp were carried out using the broodstock F0 carps, which consists of the Korean and the Chinese Israeli carps. In the spring, sexually mature breeders were selected for production of families. Selection of broodstock is based on genetic analysis and morphological analysis. Based on genetic analysis of a total of 254 broodstock, male individuals with a calculated value of the genetic relationship with females and a genetic distance value of 0.2 or less were excluded. Overall, 100 broodstock samples were selected based on morphological characteristics. Specifically, the total weight, CF, and scale score were converted into 10 points, and the breeding value was calculated by the total weight (70%), CF (20%), and scale score (10%, scale score 2 or less) for each item, i.e., breeding value (BV) = (Weight×0.7) + (CF×0.2) + (Scale×0.1). A total of four mating groups (Chinese C; ♀×Chinese C; ♂), (Chinese C; ♀×Korean K; ♂), (Korean K; ♀×Chinese C; ♂), (Korean K; ♀×Korean K; ♂) were set up to produce F1. We selected a Korean Israeli carp with mean measurement traits and set up a mating scheme to compare with common Israeli carp in Korea. According to the mating scheme, female and male were placed in each tank and spawned through water temperature stimulation (maintenance at 25℃). When egg spawning completed, eggs were collected and hatched using an artificial incubator. After 30 days of age, individuals from each group were randomly selected and raised in one tank.
Morphological analysis of F1 group
The produced F1 was analyzed from 20 to 170 days after hatching and once more at 17 months. Fish were randomly sampled from the tank, and used for growth and scale measurements and genotype analysis. The F1 group were anesthetized by immersion in 100mg/L MS-222 (Sigma, MO, USA) diluted in water. After the fin (1cm2) was taken and stored in ethanol tube (99.9%). Immediately after the measurement, they recovered from anesthesia in the recovery tank. And then, they were sent to the rearing tank for management. Length and individual mean weight were measured using the ZEN lite 2012 software program. Then, an RFID tag was inserted into each carp of the selected F1 generation offspring, and the metric trait data for each individual were collected by measuring the total length, height, weight, CF, and scale score for each unique RFID number.
DNA extraction, PCR and parentage analysis
Fin samples of F1 (1,500 samples) and broodstock F0 (100 samples) were collected and preserved in 99.9% ethanol and transferred to the laboratory for DNA extraction . Each sample was mixed with 5% Chelex 100 (Bio-Rad Laboratories, Hercules, USA) and 1% proteinase K (Roche, USA) in sterile water, incubated at 55℃ for 1 hour and then incubated at 100℃ for 10 min. The mixture was centrifuged, and the supernatant was retained. Six suitable markers were selected to distinguish diversity in carp. For PCR, we used primer pairs designed to amplify markers MFW1, MFW14, MFW16, MFW24, MFW26, and MFW30  and subjected the forward primers to fluorescent dye labeling. The following primers were used: MFW01, forward 5′-GTCCAGACTGTCATCAGGAG-3′ and reverse 5′-GAGGTGTACACTGAGTCACGC-3′; MFW14, forward 5′-CAGAAGCTTCTGGAAATCTGAG-3′and reverse 5′-GCGAGAAGATTGATGGACAAC-3′; MFW16, forward 5′-GTCCATTGTGTCAAGATAGAG-3′and reverse 5′-TCTTCATTTCAGGCTGCAAAG-3′;MFW24, forward 5′-GCTCCAGATTGCACATTATAG-3′and reverse 5′-CTACACACACGCAGAGCCTTTC-3′;MFW26, forward 5′-CCCTGAGATAGAAACCACTG-3′and reverse 5′-CACCATGCTTGGATGCAAAAG-3′;MFW30, forward 5′-GGTCAACAAGTAGTTGTGCAG-3′and reverse 5′-CCATCTCTGTCATTGCAACAG-3′. Each 20 μL reaction mixture contained 2-4 μL template DNA (100~150 ng), forward and reverse primers each 0.5 mM, 1x F-star Taq Reaction buffer (BioFact, Korea), 0.2 mM of dNTP (BioFact, Korea), 1.25 U of F-star Taq DNA polymerase (BioFact, Korea), and 1 μL dimethyl sulfoxide (Sigma St Louis, USA). Thermal cycling was conducted on an Applied Biosystems thermal cycler (#9902) using the conditions as follows: 95℃ for 2 min followed by 30 cycles of 95℃ for 30 s, 58℃ for 30 s, and 72℃ for 30 s, followed by a final extension at 72℃ for 10 min. The generated products were confirmed by 2% agarose gel electrophoresis. For fragment analysis, 1 μL of each PCR product was combined with formamide and a 500LIZ size standard (GeneScan™ 500 LIZ; Applied Biosystems, USA), and the mixture was subjected to capillary electrophoresis on an ABI 3130 DNA Sequencer (Applied Biosystems, USA). The results were analyzed using the GeneMapper version 4.0 fragment analysis software (Applied Biosystems, USA). The parentage test was performed using the PAPA 2.0 program using genotyping data.
Results were expressed as mean ± SD, and SPSS program was used for statistical significance test of all mean values for each measurement value. Significance was tested by t-test and ANOVA. The significance criterion for all P values was 0.05. The data were analyzed by one-way ANOVA using the SPSS statistical package (SPSS 5.0; SPSS Inc., USA). Means were separated using Duncan’s multiple range test and were considered significantly different at P < 0.05 .