ESTABLISHMENT OF A PATERNITY TEST METHOD FOR SELECTIVE BREEDING FAMILIES OF MEGALOBRAMA AMBLYCEPHALA

The objective of this study was to establish an accurate and economical family identification system for Megalobrama amblycephala, aimed at providing a foundation for genetic parameter evaluation and genome and molecular marker breeding of the species. Firstly, 9 pairs of “Pujiang No. 2” parents with high hypoxic tolerance and good growth performance were screened to establish 17 full-sib families. Subsequently, utilizing a “two-stage screening” process, 228 progeny exhibiting hypoxia tolerance and rapid growth were obtained. The parentage analysis of these, 228 progeny was conducted by using 8highly polymorphic microsatellite loci specific to Megalobrama amblycephala “Pujiang No. 2”. Parental and progeny DNA was amplified by a simple PCR reaction, and the resulting PCR products were separated by non-denatured polyacrylamide gel electrophoresis. With PBR322 DNA/MSPI as the marker, the size of the band alleles on the gel was determined by using Quantity One V4.6.2software. Subsequently, the genotypes of both offspring and parents were formatted into text and input into the CERVUS software for parentage analysis. This analysis primarily involved allele frequency analysis, simulation analysis and paternity analysis in CERVUS software, with paternity relationships determined based on LOD values. Allele frequency analysis showed that the average number of alleles (N_a) of the 8 microsatellite markers was 8, with an average observed heterozygosity (H_o) of 0.813, average expected heterozygosity (H_e) of 0.775, and average polymorphism information content (PIC) of 0.743. Hardy-Weinberg equilibrium testing showed that EST841, EST12, and EST158 were consistent with equilibrium, while other loci exhibited deviations. Exclusion probability analysis showed that when parental genotypes were unknown, the probability of exclusion at a single locus ranged from 0.339 to 0.540, with an average of 0.413. When one single parent genotype was known, the exclusion probability for the other of parents at a single locus ranged from 0.470 to 0.704, averaging 0.578. With both parental genotypes were known, the parent exclusion probability at a single locus ranged from 0.654 to 0.871, averaging 0.763. The cumulative exclusion probability were 98.47% (CE-1P) when both parents were unknown, and 99.99% (CE-2P) when one parent was known, and 99.99% (CE-PP) when both parents were known. Parentage analysis showed that the theoretical identification rate of 8 microsatellite loci was 99.44% when simulating 10,000 offspring and 18 parents with known parental sex. However, the actual identification rate of parents for 221 offspring was 96.93%. This study successfully utilized eight highly polymorphic microsatellite loci were used to identify the offspring of hypoxia-tolerant and fast-growth Megalobrama amblycephala screened by the two-stage method, thereby identifying parents and corresponding families for 221 (96.93%) offspring, mitigating the influence of common environmental effects in single family breeding. This accomplishment lays a foundation for further genetic parameter evaluation of hypoxic-tolerant and fast-growing families of Megalobrama amblycephala.