Identification And Application Of Hypoxia-associated Molecular Markers In Megalobrama Amblycephala

Blunt snout bream(Megalobrama amblycephala,Yih),commonly known as Wuchang fish,or bream,is an important economic herbivorous fish in China,with a total of aquacultural production more than 700,000 tons in 2015.Compared with Ctenopharyngodon idellus,Cyprinus carpio,Carassius auratus and other freshwater fish species,M.amblycephala is hypoxia-sensitive.With the continuous increasement of consumption and deterioration of aquaculture environment,it is essential for the aquaculture development of M.amblycephala to select a new variety(strain)with hypoxia-tolerance.However,hypoxia tolerance is a complex quantitative trait controlled by multi-genes,and the genes and molecular markers related with hypoxia are indistinct.Therefore,in the present study,to investigate the genetic characteristics of the hypoxia trait,we constructed base populations of hypoxia-tolerance and hypoxia sensitivity and analyzed transcriptome changes of muscle tissues between two populations based on high-throughput sequencing.Additionally,hypoxia-associated SNPs were also analyzed and verified in the progeny population.The main results and conclusions as following,1.Genetic analysis of hypoxia trait in M.amblycephalaBody length,body weight and body height were significantly different between the hypoxia-tolerant and hypoxia-sensitive groups(p < 0.05),while the head length had no significant difference(p > 0.05).Large fishes were more sensitive to hypoxia than the small ones.The hypoxia-tolerant and-sensitive base populations were established by hypoxia stress,and the heritability of hypoxia trait was 0.31(p < 0.05),a moderate heritability level.2.Characteristics analysis of muscle transcriptome under hypoxia stress of M.amblycephalaIllumina HiSeqTM2500 high-throughput sequencing platform was used to perform deep sequencing of three different hypoxia groups.A total of 96,934,854 raw reads and 88,200,889 clean reads were generated,with a GC content of about 50%.A total of 57,303 Unigenes(mean length = 807 bp,range = 201-21643 bp)were obtained after assembly,and the N50 and N90 values were 1465 bp and 298 bp,respectively.A total of 426 differentially expressed genes(DEGs)were identified.Overall,the number of down-regulated genes was higher than that of up-regulated genes under hypoxia stress.The biological function enrichment of down-regulated differential genes are related to the assembly and organization of protein-macromolecular complexes,and the up-regulated differential genes are mainly involved in muscle contraction and blood circulation.GO enrichment of DEGs among treatments and the mechanism of biology process in muscle tissue under hypoxia stress could be deduced.The main purpose of muscle tissue to deal with hypoxia is to maintain low energy levels,and differences in the ability to control metabolic energy requirements could be the potential functional consequences of the transcriptome variation.3.Screening and development of hypoxia-associated SNP markers64,797,236 bp were sequenced.52,623 probable SNPs were detected.The transition and transversion types of SNPs were 30192 and 16802 respectively,with a ratio of 1.8.The synonymous SNPs accounted for 99.7% and non-synonymous only 0.3%.Twenty eight non-synonymous SNPs of 16 genes were selected for subsequent experiments and five polymorphic SNPs were identified using polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP)method.Polymorphism information content(PIC)was 0.1436-0.3469 with the average of 0.2669.In addition to Nr4a1-C2188 T,the other four SNPs were in line with Hard-Weinberg equilibrium and 2 SNPs(Plin2-A1157 G and Hif-3a-2917AG)had significant frequency differences between the hypoxia-tolerant and-sensitive populations(p < 0.05).4.Hypoxia performance test of offspring population and validation of hypoxia-associated SNP markersThere was no significant difference in fertilization and hatching rate among progenies of different hypoxia-tolerant parents(p>0.05).The offsprings of hypoxia tolerant-parents had the strongest tolerance to hypoxia,and those of hypoxia susceptible-parents were the weakest.The most tolerant group to hypoxia has the smallest weight,and the weakest has the largest.The relationships between Plin2-A1157 G and Hif-3a-2917 AG,and hypoxia trait were not significant in all offspring populations.