Establishment Of Hybrid Strains In Closely Related Tilapines And Screening Of Sex-linked Markers

Tilapia,a common name of a group of cichlid fishes native to Africa and Middle East,is the second most cultured freshwater species group worldwide.Tilapia exhibit sexual dimorphism in range of traits of interest like growth,body size or age at maturity.Research on the sex determination mechanism of tilapia has been a difficult and hot topic in breeding research,driven by an aquacultural interest in stocking of all-male fingerlings,which is not only because males grow faster that greatly improve growth rates and overall production but also to avoid uncontrolled reproduction before harvest.Variation in the genetic mechanisms of sex determination was found among tilapia species,among which there are paternal heterogametic(XX-XY)species,including Nile tilapia and Mozambique tilapia,and maternal heterogametic(ZZ-ZW)species,including blue tilapia.Even in the same species,the sex determination mechanism is complicated.Some Nile tilapia strains have an XY system on linkage group(LG)1,while others segregate an XY system on LG23.The closely related blue tilapia segregates both an XY locus on LG1,and an epistatically dominant ZW locus on LG3.Nile tilapia,blue tilapia and Mozambique tilapia are closely related species of the Oreochromis species.Different cross combinations usually resulted in male biased sex ratios,heterosis and fertility through interspecific crosses.However,in some cross combinations,there was inconsistency in obtaining all-male offspring,which may result from using impure/contaminated stocks of one or both strains.It is an effective way to solve this problem via molecular marker assisted breeding by screening sex-linked marker and cloning sex-determining genes for genetic manipulation.Establishment of hybrid strains and near-isogenic lines are helpful to screen the sex-linked marker,and lay the foundation for positional cloning of sex determining gene.Moreover,it is of great significance to produce all-male fingerlings in aquaculture and clarify the mechanism of sex determination.In this study,the Nile tilapia,blue tilapia and red tilapia(hybrids between Nile tilapia and Mozambique tilapia),were chosen as experimental animals to establish hybrid strains.Previous study suggested there is the sex determining region located on the LG1 for tilapia.The Z/Y/X sex-chromosome markers were identified and the putative sex determining region on LG1 was narrowed based on whole genome re-sequencing analysis of blue tilapia using genome assembly of Nile tilapia as the reference.The main results are as follows:1.A near-isogenic line was established from the XX(Nile tilapia)female and the ZZ(blue tilapia)male.The F1 hybrid males(ZX)were backcross(BC)with the maternal XX female.The progeny produced by the first backcross was denoted as BC1.Next,the XX female were crossed with BC1 male.By repeatedy backcrossing,hence,the sex determining region of all males of the backcross generation was derived from the ZZ while the genetic background from the maternal XX female.So far,we have obtained the BC3.The F1 hybrids ZX were all-male,and the sex ratio from progeny of backcross was 1:1.Crossing of F1 male(ZX ♂)and female from a sex-reversed progeny(ZX ♀)to generate genotype XX : ZX : ZZ = 1:2:1 and 1:3 female-to-male broods.In addition,transition of the sex determination system in blue tilapia was achieved by the crosses.The sex determining locus of hybrids are located neither on LG23 of Nile tilapia nor on LG3 of blue tilapia,but rather located on LG1,which demonstrated that interspecific hybridization lead to the transition of sex chromosomes.2.The structural variations with large sequence difference on LG1 were obtained via comparing the ZZ genome sequence of blue tilapia(obtained by re-sequencing)with XX reference genome of Nile tilapia.Then the structural variations were converted into sequence characterized amplified regions(SCAR)markers.Totally,200 markers were screened,among which 27 SCAR markers could distinguish the genotypes ZZ,ZX and XX of the hybrid progenies.These markers were named from Marker-1 to Marker-27,and used to identify the genetic sex and recombinant individuals of all offspring.Based on the screening results of recombinants in 73 individuals of BC1 population,the genetic linkage map was constructed using JoinMap 4.0.The recombination rates of Marker-22,-23,and-24 were the lowest among all of the 27 SCAR markers,and they supposed most closely linked to sex determining locus.Marker-22 was found to have the highest association phenotypic sex among all the markers because it given accuracy of 96.7%.Followed by a series of markers between Marker-10 and Marker-21 with the same accuracy of 96.2%.Marker-23 was the third highest association with an accuracy of 96.0%.Taken together,the sex determination region was located on LG1 between Marker-10 to Marker-23,and the corresponding genomic physical distance is supposed about 0.6 Mb.3.A hybrid strain was established from Nile tilapia females(XX)and the red tilapia males(XY).The cross resulted in 1:1 sex ratios.The F1 hybrids display coloration pattern of red skin background with scattered black spots.A pair of F1 hybrid female(XX)and male(XY)were selected to generate an F2 family.The F2 hybrids display the segregation of coloration pattern of 3 red : 1 black,while the sex ratio was 1:1.This suggerted that red is controlled by an autosomal dominant single gene and is not linked to sex.4.The sex chromosomes of F2 hybrids red tilapia were also located on LG1.Selecting of 17 sex-linked markers from the blue-Nile hybrids were also applicable for the red-Nile hybrids.The remaining 10 markers were proved as polymorphic sites in this hybrid strain.The 17 sex-linked markers identified 10 recombinant individuals in the 120 individuals of F2 generation,of which 9 display female phenotypic but with male genotypic.Based on the above results,the sex-determining region of the red-Nile hybrid fish was supposed covers the above 17 sex-linked markers,and the Marker-10 to Marker-23 region overlaps with the sex-determining region of the blue-Nile hybrid fish,indicating the sex determination region on LG1 was most likely located on the same overlapped region.The results of the two hybrid strains were slightly different,suggesting that even in the case of closely related species hybridization,due to differences of parents in the sex determination system,sex chromosome,and sex determination genes.The hybrids progeny display differences regions of recombination suppression.In summary,tilapias are a well-suited model species for the analysis of the gene network controlling sex determination and the early events in sex chromosome evolution.Using hybrid strains of closely related tilapines,the transition between different sex determination system and sex chromosome can be achieved by hybridization.Moreover,the same sex determining region above on LG1 in different hybrid strains due to rcombination suppression,indicating that this region is conserved in different tilapias,and may be the ancestral state of LG1.This results is helpful to understand of the molecular mechanism of sex determination,the evolution of sex chromosomes,and the adaptive radiation,rapid speciation and biodiversity of African cichlid fishes.Furthermore,it is easier to obtain molecular markers through hybridization,which lays the foundation for positional cloning the sex determining gene,and has broad application prospects in producing all-male fishes and molecular marker-assisted breeding of tilapia for aquaculture.