Development And Application Of Whole-genome Microsatellite Markers In The Pufferfish

Takifugu fasciatus,commonly known as river pufferfish,is widely distributed in the middle and lower reaches of the Yangtze River,the Yellow Sea and the Bohai Sea.It is an aquaculture species with high economic value.In recent years,with the continuous expansion of T.fasciatus breeding industry,problems(such as environments pollution and inbreeding)have caused serious decline in the pufferfish germplasm resources.Therefore,it is urgently needed to develop efficient and practical molecular markers to improve the germplasm of pufferfish.At present,there are fewer molecular markers developed or available for T.fasciatus,which has become one of the main "bottlenecks" restricting the molecular marker-assisted breeding of T.fasciatus.In this study,we firstly analyzed the distribution characteristics of microsatellites(also called simple sequence repeat,SSR)in the whole genome of T.fasciatus.Then we developed the microsatellite primers of T.fasciatus in batches.Finally,we identified the microsatellite markers related to the growth traits of T.fasciatus,using which we further distinguished the species of T.fasciatus,Takifugu rubripes and hybrid pufferfish(T.fasciatus♀×T.rubripes♂)and analyzied their genetic diversity.Our study provides a resource for the work on the hybrid breeding and molecular marker-assisted breeding of T.fasciatus and other Takifugu populations in the future.The main findings of our study are as follows:1.Distribution characteristics of microsatellites in the whole genome of T.fasciatusIn this study,using the second-generation genome sequencing data of T.fasciatus,MISA software was used to screen and analyze the microsatellites in the whole genome of T.fasciatus.The results are as follows: A total of 137293 complete microsatellites were obtained from the genome of T.fasciatus containing approximately 365 Mb.The total length of microsatellites is 2868151 bp,accounting for 0.78% of the total length of the genome sequence,and the relative abundance is375 bp/Mb.Among the 1 to 6 base type microsatellites,the number of mononucleotide type is the largest,accounting for 47.72% of the total number of complete microsatellites,followed by dinucleotide(38.64%),trinucleotide(7.45%),tetranucleotide(4.25%),pentanucleotide(1.44%)and hexanucleotide type(0.51%).The changing trends of microsatellites of different repeated copy types are generally consistent.As the copy number of the core sequence increases,the number of microsatellites decreases.In addition to the obvious differences in the number distribution of the six types of microsatellites,the distribution of microsatellites in different categories of the same base type is also very different.The 10 categories of microsatellites with the largest number of repeats in the genome of T.fasciatus are:AC,A,C,AG,GAG,AT,AAT,AGAGG,GGC and AAC,which added up to 125617,accounting for 91.50% of the total number of complete microsatellites.2.Screening of microsatellite markers related to growth traits of T.fasciatusFirst,using the primer design software Primer 5.0,a large number of microsatellite primers were developed in batches from the whole genome of T.fasciatus,and then 100 pairs were randomly selected for quality inspection,and 42 pairs of microsatellite primers with polymorphism were selected.The chi-square test was used to analyze the differences of genotype distribution of 42 microsatellite loci in the large and small populations of T.fasciatus.Seven microsatellite loci with significant differences were obtained,which were used to analyze the correlation between genotype and multiple growth traits of a random population of T.fasciatus.The results show that there are 5 microsatellite loci with different genotypes that are significantly correlated with multiple growth traits of T.fasciatus.Among them,AC at XH11 is the inferior genotype;AB at XH63 is the dominant genotype,and BB is the inferior genotype;XH94 at CC is the dominant genotype;BC and CD at XH96 are the dominant genotypes,AB,CC and BG are inferior genotypes.The microsatellite markers correlated with the growth traits of T.fasciatus obtained in this study could effectively assist the selection of superior parents.3.Identification of T.fasciatus,T.rubripes and hybrid pufferfish(T.fasciatus♀×T.rubripes♂),and comparison of population genetic diversityOne hundred pairs of microsatellite primers developed and randomly selected from the whole genome of T.fasciatus were used for PCR amplifications in T.fasciatus,T.rubripes and hybrid pufferfish.The microsatellite marker XH77 was successfully screened.After testing,the amplified products of this marker can produce obvious different bands in the three Takifugu(3 bands were amplified from T.fasciatus,and the positions were about 150 bp,800 bp and 1500 bp respectively;3bands were amplified from T.rubripes).The positions of the bands are about 150 bp,1000 bp and 1800 bp;hybrid pufferfish amplified 5 bands,the positions are about 150 bp,800 bp,1000 bp,1500 bp and 1800 bp).The accuracy rate of identification is100%.Twenty-one pairs of microsatellite primers were used to analyze genetic diversity and genetic relationships among three Takifugu populations.The result shows that the average number of alleles of the T.fasciatus,T.rubripes and hybrid pufferfish population are 7,6 and 7,respectively;the average effective allele numbers are 4.271,3.000 and 3.979;the average observed heterozygosities are 0.691,0.596 and 0.673;the average expected heterozygosities are 0.720,0.595 and 0.719,and the average polymorphic information contents(PIC)are 0.668,0.542 and 0.672,respectively.The genetic information richness of the population of hybrid pufferfish is similar to that of T.fasciatus,and hybrid pufferfish has higher genetic information polymorphism.The population of T.rubripes has the lowest genetic diversity.The genetic distance between T.fasciatus and hybrid pufferfish(1.135)is smaller than that between T.rubripes and hybrid pufferfish(1.185),while the genetic distance between T.fasciatus and T.rubripes(1.843)is the largest.The clustering results of the phylogenetic tree showed that the hybrid pufferfish and the T.fasciatus were clustered firstly,then the two were clustered with T.rubripes.The above genetic information shows that the relationship between hybrid pufferfish and the female parent T.fasciatus is closer,which indicates the maternal inheritance.