Genetic analysis of key genes Spo11 and Mlh1 controlling meiosis in hybrid fish of Carassius auratus red var.(♀)×Cyprinus carpio L.(♂)

【Objective】 The present study analyzed the characteristics of gene sequences and expression of key genes (Spo11 and Mlh1) in meiosis from the hybrid crucian carp(Carassius auratus red var., ♀, abbreviated as RCC)×common carp(Cyprinus carpio L., ♂, abbreviated as CC), which would reveal whether there was selection pressure for meiosis factors in hybrid lines, providing the genetic basis for understanding the mechanism of breaking through reproductive barriers in hybrid fishes.【Method】 In this study, the hybrid strains of RCC×CC which has formed stable strain was taken as research objects, containing RCC, CC, allodiploid hybrid of RCC(♀)×CC(♂) (abbreviated as F₁) and allotetraploid hybrid of RCC(♀)×CC(♂) (abbreviated as 4nAT), respectively. The genetic and expression characteristics of Spo11 gene and Mlh1 gene in hybrid crucian carp were studied by molecular cloning, sequence alignment and Western blot, and the gene-tic relationships among them and their original parents were analyzed.【Result】 Most of the CDS sequences of Spo11 gene in RCC, CC, F₁ and 4nAT were 1152 bp, butsome1157bp CDS sequences were also found in 4nAT. Two independent genetic fragments of the original parents were stably obtained in F₁ and 4nAT, and the recombinant gene fragment ［4N-SPO11-11-2(MT648223) ］of the original parents was also detected in 4nAT, and its similarity to the corresponding fragment of the RCC-SPO11 gene of the maternal was 96.2%, and to the corresponding fragment of the CC-Spo11 gene of the paternal was 93.7%, indicating that the expression pattern of Spo11 gene was not completely conserved and 4nAT possessed some genetic variations. CDS sequences of Mlh1 gene also were stably in hybrid crucian carp, showing stable heterozygous genetic characteristics, namely both CDS sequences of RCC-MLH1 gene(99.5%) and CC-MLH1 gene (99.4%) in F₁ and 4nAT, and there were only a few gene loci variation in the corresponding genetic fragment between the original parents and hybrid fish. Furthermore, the results of western blotting showed that both SPO11protein and MLH1 protein expressed normally in the hybrid progeny of RCC(♀)×CC(♂). The SPO11-β subunit with large molecular weight was only expressed in the testis of 4nAT, while SPO11-α subunit with small molecular weight was only expressed in testis of F₁.【Conclusion】 These results show that F₁ and 4nAT stabilize the Spo11 gene and Mlh1 gene of the heterozygous inherited original parental RCC, although there are small differences in CDS sequence structure, they can express related protein independently. It indicates that key meiotic genes are expressed normally during hybrid fish formation, and lay an important genetic basis for the analysis of the distant hybridization to overcome reproductive barriers.