| The Quasipaa spinosa had the characteristics of sweet meat,delicious taste,rich nutrition,and high economic and scientific research value,so it had attracted more and more people’s attention.In order to meet the market demand and protect wild resources,it had become an inevitable development trend to carry out artificial breeding of Q.spinosa.However,in the past,the mode of wild fishing and in-situ breeding was mainly adopted,and the prevention and control and research on population genetic diversity were lacking,resulting in serious inbreeding of breeding population,poor disease resistance and small size of Q.spinosa.With the increasingly mature artificial breeding technology of Q.spinosa,germplasm innovation had become an important guarantee for the sustainable development of large-scale breeding industry of Q.spinosa.Using appropriate molecular markers for genetic diversity research or breeding of improved varieties could effectively promote the development and innovative utilization of germplasm resources.Therefore,in this study,the breeding population of Q.spinosa was used as the experimental object,and the genetic diversity of Q.spinosa breeding population was evaluated from the nuclear and cytoplasmic levels by transcriptome sequencing technology,microsatellite loci based on transcriptome development technology,mitochondrial COI gene cloning and genetic analysis,in order to solve the current situation of breeding benefit reduction caused by the decrease of genetic diversity in the process of artificial breeding of Q.spinosa.1.SSR polymorphic feature analysis based on Q.spinosa transcriptomeA total of 81.74 Gb Clean Data was obtained by the transcriptome sequencing and database of Q.spinosa.93,887 Unigenes were obtained from the head splicing assembly.The transcriptome GO、COG and KEGG databases were analyzed.It was found that there were more genes involved in the regulation of the immune system,which provided a research direction for explaining the important drug and food supplement effect of Q.spinosa.Totally,33,019 potential SSR markers were identified with 21,966 unigenes harboured only one SSR.In additional,a total of 6688 unigenes have more than one SSR locus.Di-nucleotide(25,788)and Tri-nucleotide(4807)are the most abundant motif type while A/T(83.88%)and AC/GT(38.15%)are respective the most frequent di-,tri-nucleotide repeats.Among the 33,019 potential SSR markers,the times of repetition is mainly between 5-25 times.In additional,there are only 1633 were located in the coding area.Statistics got from repetitive sequence length more than 12 bp SSR markers reach to 17,244 accounting for 58.53% of all the SSRs,it was very rich polymorphism.The result indicated that the Q.spinosa has a middle to high level of genetic diversity.2.Genetic diversity analysis among cultured populations of Q.spinosa based on microsatellite lociThe SSR loci found in the transcriptome were designed according to the principle of removing single base repeats and complex repeat types,and the sequences with length ≥ 50 bp at both ends of SSR were selected for primer design.250 primers were randomly selected for SSR loci verification.It was found that 100 pairs of primers amplified a single band,and the size of the band was consistent with the expectation.The amplified products were observed by 8% non-denaturing polyacrylamide electrophoresis and silver nitrate staining.According to the stability of bands,20 microsatellite markers were chosen to comprehend the genetic information based on148 samples from five breeding populations(Jiangxi,Zhejiang,Guangxi,Guangdong,Fujian).A total of 135 different alleles were detected,with mean number of alleles and effective alleles were 6.75 and 3.085,respectively.The observed mean heterozygosity was 0.479,and the expected heterozygosity was 0.637 while the polymorphic information content reached to 0.591.Among populations,the heterozygosity and expected heterozygosity were ranged from 0.385 to 0.518 and 0.463 to 0.561,respectively,with the heterozygosity is lesser than expected heterozygosity.The genetic relationship of Guangdong and Guangxi population showed the highest genetic similarity(0.841),while Fujian and Jiangxi population showed the lowest genetic similarity(0.524),according to the reslut based on UPGMA clustering.The study indicates that the cultured population of Q.spinosa has a degree inbreeding,suggesting that the breeding range should be expanded or introduce superior varieties from other fields as breeding parents,regularly,to reduce inbreeding.3.Genetic diversity analysis among cultured populations of Q.spinosa based on mitochondrial COIIn 5 breeding populations,COI gene was used for genetic diversity analysis.A total of 60 haplotypes were defined,and each haplotype was unique.Zhejiang population had the largest number of haplotypes,while Guangdong population was the least.The genetic diversity of breeding populations was high except Guangxi and Fujian populations,and the three populations were low.There was a great differentiation among populations,and the population variation mainly came from among populations(67.05 %).Comprehensive analysis found that the genetic diversity of Q.spinosa breeding population decreased,the analysis may be due to the phenomenon of inbreeding in artificial breeding population,and the founder effect or artificial selection,resulting in the decline of genetic diversity of artificial breeding population.In summary,the genetic diversity of Q.spinosa breeding population showed a downward trend.Therefore,active measures should be taken to improve its genetic diversity during the breeding process,expanding the selection of individuals for mating at greater distances;regularly replenish individuals from different populations in different regions as back-up parents to enhance inter-regional exchange;to conserve high quality germplasm resources of Q.spinosa and realize sustainable utilization of resources. |
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