Linkage Map Construction And Mechanisms For Sex Determination And Shell Color In The Pacific Abalone(Haliotis Discus Hannai)

The Pacific abalone(Haliotis discus hannai)is one of the most economically important mollusk species in China.Since its artificial cultivation started in the late1980s,selective breeding and crossbreeding have greatly accelerated the abalone industry.However,the shortcomings of the traditional breeding methods,such as the longtime period,low accuracy,low efficiency in traits with low heredity,have restricted the further development.With the help of molecular markers and genomic tools,the process of selective breeding can be stimulated significantly.In this study,linkage maps were constructed using both microsatellites and SNPs developed through genotyping-by-sequencing(GBS).Based on the maps,mechanisms controlling two traits(i.e.,sex and shell color)in the Pacific abalone were demonstrated.The main progresses are as follows:1.Based on the existing microsatellites,integrated linkage maps of the Pacific abalone were constructed using three F₁ families.A total of 352 markers were successfully located on the map.Both the female and male map contain 18 linkage groups,which are consistent with the chromosome number of the Pacific abalone.The female map contained 273 markers,spanning 927.3 c M,with the average distance of3.64 c M.The male map consisted 277 markers,spanning 727.0 c M,with the average distance of 2.80 c M.According to the constructed maps,we analyzed the sex-specific recombination.It showed that the total length of the male maps were significantly shorter than that of the females'in all of the three mapping families,and the ratios of the cumulative genetic distance between the female and the male map were 1.66,1.82and 1.23 respectively,suggesting a male biased recombination suppression.Moreover,site-specific recombination differences between female and male were observed along the chromosomes.Segregation distortion markers were analyzed for each mapping family.The results suggested that in family GL₉₈O,71.43%of the distortion were caused by homozygote deficiency,however,in family#15 and family GG₃,the distortion mainly resulted from heterozygote deficiency,which explained 55%and 60%of the total distorted markers.The revised linkage map based on SSR provides framework for QTL mapping and MAS in the Pacific abalone.2.High-density linkage map was constructed based on the GBS technology.Mapped 7,082 markers,the female map spanned 1884.39 c M,mapped 7,082 markers,with an average interval of 0.27 c M,while the total length of the male map was 1555.67c M,with an average interval of 0.34 c M,and 4,624 markers were located.The integrated map contained 10,704 markers,spanning 1652.75 c M,with an average interval of 0.15 c M.Construction of high-density linkage map supplies solid foundation for the subsequent analyses,including fine mapping for the target traits,genomic selection and the genome assembly.Besides,a total of 1,637,993 SNPs with read depth>12×in parents were detected,providing a sound basis for marker development on the genomic scale in the Pacific abalone.3.Sex determination system in the Pacific abalone was deduced according to the phenotypic sex and the genotypes of polymorphic markers in family GL₉₈O.After testing the 176 segregating markers,only 8 markers were significantly associated with sex(P<0.00001),which were all located on LG3.Among them,Afa203 was completely linked to phenotypic sex,and the alleles of Afa203 in the sire were sex-specific inherited.Thus,the sex determining system in the Pacific abalone is male heterogamety(female:XX;male:XY).Applying both linkage and association analysis,sex-determining QTL was mapped to the 54.861 c M of LG13.Furthermore,to make full use of the re-sequencing dataset,a bulked segregant analysis(BSA)approach was adopted,with female and male individuals of the highest read depths chosen to construct pools of different sex.The ratios of read depths in all markers were calculated between the two pools.It showed that 75.15%of the most associated markers distributed mainly on 29 scaffolds.Furthermore,genotypes of these markers were deduced and corrected according to the adjacent markers located on the same scaffold.Then,R package GWAF was used to analysis the linkage between these markers and the phenotypic sex,and makers on 16 scaffolds were detected to be completely linked.4.Mechanism determining the shell color of the Pacific abalone was demonstrated,and the locus controlling green/orange shell color was further mapped.In family#15,three shell color morphs(green,orange,blue)were exhibited in the progeny.By taking both“one-locus”and“two-locus”controlling model into consideration,goodness-of-fit tests proved that the shell color in the Pacific abalone was controlled by a simple recessive epistasis model.In order to map the locus determining green/orange shell color,both linkage and association analysis were conducted,and the peak value was observed at 73.103 c M of LG4.Moreover,BSA analysis was performed to find the markers most associated with shell color.Green-shelled and orange-shelled individuals with highest read depths were chosen to form pools of distinct shell colors.By calculating the ratios of read depths in all markers,those most associated with shell color were detected.It showed that 75%of the most associated markers came from six scaffolds.After genotype deduction and correction,GWAF was used to confirm the linkage between these markers and shell color.However,no completely linked markers were detected,and the most linked markers located on five scaffolds still showed three recombinants.