| Grass carp(Ctenopharyngodon idella),the most productive aquaculture species in China,was great importance for Chinese aquaculture industry.It was of great theoretical and practical significance to study on molecular marker-assisted breeding of grass carp.Previous studies had developed microsatellite markers,also called simple sequence repeat(SSR),applying in breeding of growth and disease resistance traits by enrichment library method.However,these SSR markers were generally based on 2 base repeat sequences,which were prone to misjudfment in genotype interpretation.The accuracy is affected in the actual use of parentage identification of grass carp.At present,the whole-genome of grass carp had been published,which provided possibility for developing 4-6 base repeat SSR with high polymorphism.In addition,the breeding program on grass carp were focused on growth and disease resistance.However,the existence of intermuscular bones(IBs)seriously affects the eatability and processability of grass carp which resulting in a decrease in economic value.Previous study found natural mutants of grass carp without IBs which had no affect to growth and activity.This result showed possible to cultivated new varieties of grass carp with little or without IBs.The existing studies on IBs of grass carp remain in morphological and the previously developed SSR markers have certain defects in use.Therefore,this study explored the SSR loci based on the whole-genome data of grass carp and a variety of fish were analyzed accordingly.The highly polymorphic SSR markers were developed and used to parentage analysis for grass carp.The genetic parameters of IBs number were evaluated in grass carp to explore the feasibility of selective breeding of IBs number trait.The main results are as follows:1.The analysis of SSR characteristics of 67 fish species based on the whole genome sequencing dataMISA software was used to develop the SSR loci from 67 species genome data including grass carp and the characteristics of SSR loci in these species were analyzed.The results showed that the genome size of 67 species of fish was 342.40 Mb-2966.89Mb,including 160,420-2,764,367 SSRs with average of 698,242.24.The proportion of SSRs was 0.26%-6.17%with average of 1.57%.In 12 species of Cypriniformes,we found that the number of SSRs was positively correlated with genome size(R~2=0.8742,p<0.001).There was significant difference in the number of different repeat types SSRs.The number of 1 base or 2 base repeat SSRs were the most frequently and 5 base or 6 base repeat SSRs were the minimum among 67 species.The dominant repeat units from 1-3 base repeat SSRs were relatively fixed in most of fishes,which were A/T,AC/GT,AAT/ATT.The 4-5 base repeat SSRs were significantly different among these species,but in the three species of Anguilla(AAAT/ATTT,AATGT/ACATT),12 species of Cypriniformes(AGAT/ATCT,AATAT/ATATT),and 6 species of Medeka(ATCC/ATGG,AATAG/ATTCT)showed the consistency.The 6 base repeat SSRs didn’t find obvious regularity among these 67species.The results showed that 677,363 SSR loci are found in the 900.51 Mb genome sequence of C.idella,with a total length of 12,835,407 bp,accounting for 1.43%of the whole genome length.The average distance of SSR is 1,329.43 bp.The mononucleotide repeats motifs(52.85%)are the most common,following dinucleotide repeat(31.44%),tetranucleotide repeat(8.05%),trinucleotide repeat(6.47%),pentanucleotide repeat(1.12%)and hexanucleotide repeat(0.07%).The preponderance motifs of 1-6 base repeat are A/T,AC/GT,AAT/ATT,AGAT/ATCT,AATAT/ATATT and AACCCT/AGGGTT,respectively.2.Development of highly polymorphic SSR markers and establishment of parentage assignment platform for C.idella based on high polymorphism microsatellitesThe 110 loci with 4-6 base repeats were randomly selected and primers were designed.Among these loci,50 loci were amplified successfully with mixed DNA of C.idella used as template and 15 loci showed high polymorphsim when analyzed in its breeding population.Cervus 3.0 analysis results showed that average observation heterozygosity(Ho)of 15 loci was 0.708-0.995 and the polymorphic information content(PIC)was 0.785-0.955.The accuracy of simulated paternity test was 100%with 95%confidence degree.These 15 loci were used to identify 192 offspring from20 families.The Ho of 15 loci was 0.842-0.991;the PIC was 0.857-0.939;and all the offspring matched to the correct parents successfully with the accuracy of identification being 100%.The results of P-LOCI analysis indicated that with the increase of microsatellite loci,the success rate of paternity test gradually increased.When the number of loci reached four(C.I.4-14,C.I.4-3,C.I.5-12,C.I.5-8),the accuracy of paternity test reached 99.78%.The accuracy was infinitely closed to100%when the loci number increased.3.Evaluation of genetic parameters for IBs number trait in C.IdellaA total of 136 parents(66 males,70 females)of C.idella were randomly divided in the three groups for natural mating.The 538 offspring were selected randomly to measure growth traits,and the number of IBs was counted by boiling method.Total of190 full-sib families(58 paternal half-sib families and 56 maternal half-sib families)were identified based on six SSRs loci pedigree analysis.The IBs number was counted individually and the genetic parameters of IBs number were estimated.The animal models were used to evaluate the heritability of tested traits.The results showed that the IBs number of whole fish body was ranged from 102-127 with the average of 117,coefficient of variation(CV)being 3.22%.The CV of IBs number in ventral section(5.52%)was significantly higher than that of dorsal section(2.52%).The heritability of IBs number in C.idella showed medium level(0.293)for whole body and dorsal section had the lower heritability of 0.150 compared to that of ventral section(0.261).The results also found a high genetic correlation(0.899,p=0.041)between body height and IBs number,which may provide a new reference index for future breeding research on IBs number trait.In this study,the genome-wide microsatellite loci of 67 species of fish including C.idella were excavated to explore their quantity and distribution characteristics.Based on the genome-wide SSR loci screening for C.idella,15 microsatellite loci with high polymorphism of 4-6 base repeats were developed,and the parentage identification platform of C.idella was established.The IBs number of C.idella was counted,and the genetic parameters were evaluated to analyze the feasibility of breeding.The results of this study will provide a theoretical basis and useful tool for the subsequent selection breeding of IBs number in C.idella which support the breeding of new varieties with less IBs in C.idella. |
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