Study On The Genetic Diversity Of The Field Population Of Brachionus Calycifloruss

Cyclically parthenogenetic Brachionus calyciflorus is important species in freshwater ecosystems and an important model organism in population genetic structure research.The life cycle includes two stages of parthenogenesis and sexual reproduction.When the population was just established,a large number of dormant eggs hatched from the sediment in the water body,and the non-mixed females were hatched If the water environment factors(such as too high temperature or low oxygen content,etc.)are not suitable,the population begins to reproduce sexually and produces dormant eggs which sink into the water sediment to ensure the continuation of the population.Studies have shown that the population of B.calyciflorus is composed of cryptic species complex and it is generally believed that in field water body,there is high genotypic diversity when the population of periodic parthenogenetic rotifers is first established.However,the parthenogenesis is influenced by clonal selection,and the genotypic diversity of the population decreases with the change of seasons.Due to the life history of periodic parthenogenetic rotifers,inbreeding decline is inevitable in both wild and laboratory populations,and Inbreeding Avoidance Strategy is the evolutionary driving force of this unique mating system.However,little attention has been paid to the genotypic diversity and inbreeding decline of periodic parthenogenesis,and little work has been done in China.Based on the internal transcribed spacer(ITS)and microsatellite markers,this study was conducted in B.calyciflorus from Mingze Lake,Dalian Children's Park.The following questions were explored:(1)whether there was a cryptic species complex in small artificial water body(2)whether B.calyciflorus experienced clonal selection effect and inbreeding depression in field water body(3)the mutation rate of microsatellite sites in the B.calyciflorus.The main findings are as follows:1.Based on the experimental population of B.calyciflorus in Mingze Lake,a total of 1141 potential polymorphic microsatellite loci were obtained by transcriptome sequencing.The polymorphic level of 150 microsatellite loci was analyzed,and 22 of them were found to be monomorphic,17 of two kinds of polymorphic,7 of three kinds of polymorphic,5 of four kinds of polymorphic,3 of five kinds of polymorphic.At the same time,combined with the published microsatellite loci(12 loci)of B.calyciflorus with high polymorphism,7 microsatellite loci were used to analyze the genetic structure and microsatellite loci mutation rate of B.calyciflorus in the field population.2.In order to determine the composition of the cryptic species complex of B.calyciflorus in Mingze Lake,the phylogenetic tree of B.calyciflorus was constructed and by comparing the differences of ITS gene sequences(4% were different cryptic species).It was found that the cryptic species complex of B.calyciflorus in Mingze Lake was composed of Clade 1 and Clade 2.According to the international classification standard of B.calyciflorus,Clade 1 in Mingze Lake is classified as D and Latin name B.fernandoi sp.nov.,Clade2 is a cryptic species C and Latin name B.calyciflorus s.s..To facilitate the description of the follow-up results,Clade1 is defined as BC-D and Clade2 as BC-C.Since BC-D was less temperature tolerant than BC-C,it was replaced by BC-C after a period of early spring.3.The genetic diversity of BC-D and BC-C was analyzed using seven microsatellite markers.The results showed that the observed and effective allele numbers of BC-D population were 4.9286 and 2.8138,respectively.The observed allele number and effective allele number of BC-C population were 3.4078 and 2.2552 respectively.The observed and expected heterozygosity were 0.6077 and 0.6123,0.6918 and 0.5203 respectively in BC-D and BC-C populations,and Nei's expected heterozygosity was 0.5932 and 0.5123,respectively.The results showed that the genetic diversity of B.calyciflorus in the field was high.However,Hardy-Weinberg Genetic Balance Test showed that there were at least 2 deviations from Hardy-Weinberg balance in each population.The Genetic Deviation Index D indicates that the population is in the state of excess heterozygotes.The deviation of Hardy-Weinberg equilibrium may be caused by excess heterozygotes.The frequency variation of five rare alleles in B.calyciflorus showed inbreeding degeneration to some extent.The genotypic diversity analysis showed that the population of B.calyciflorus had high genotypic diversity and experienced clonal selection along with the succession of the population.4.In order to determine the excess of heterozygous population of B.calyciflorus in the field,we conducted a related study on the mutation rate of microsatellite loci in B.calyciflorus.In this study,the data of four microsatellite loci in four clonal populations of B.calyciflorus and cultured in the laboratory were analyzed,and the mutation rate of microsatellite loci in four clonal populations was calculated.The results showed that the average mutation rate of each microsatellite locus in the 30 th generation was between 8.25×10-4—3.33×10-2,and the average mutation rate of each microsatellite locus was 1.37×10-2.In the 50 th generation,the average mutation rate of each microsatellite locus was between 0 and 2×10-2,and the average mutation rate of each microsatellite locus was 4.65×10-3.Compared with other studies,the mutation rate of the microsatellite locus of B.calyciflorus is at a relatively high level.By comparing the allelic frequency distributions of the B.calyciflorus population and the four clonal populations in the laboratory,predation stress promoted the production of rare alleles.Moreover,the genetic structure showed a heterozygous excess state,indicating that the heterozygote excess is caused by the higher mutation rate of the microsatellite loci.In conclusion,the population of B.calyciflorus in Mingze Lake of Dalian Children's Park is composed of non-overlapping cryptic species BC-C(B.calyciflorus s.s.)and BC-D(B.fernandoi sp.nov.).Both populations had high genetic diversity.At the same time,BC-D disappeared in the water body under the pressure of temperature and cysticercosis.However,BC-D continued to breed because of the replacement of the resting egg bank.The genetic results of BC-C showed that the population experienced clonal selection effect and inbreeding depression to some extent.Under predation pressure and high mutation rate of microsatellite loci,the population of B.calyciflorus tended to parthenogenesis while producing new alleles,resulting in excess heterozygotes and deviation from Hardy-Weinberg equilibrium.