Study on the Genetic Diversity of the Field Population of Brachionus Calycifloruss

张吉

殷旭旺

硕士

大连海洋大学

萼花臂尾轮虫;;ITS;;微卫星;;遗传多样性;;突变率

周期性孤雌生殖的萼花臂尾轮虫(Brachionus calyciflorus)是淡水生态系统中浮游动物的重要种类,并且是群体遗传结构研究中常见的模式生物。生活史包括孤雌生殖和有性生殖两个阶段,种群刚建立是水体沉积物中大量休眠卵孵化出非混交雌体进行孤雌生殖快速扩增种群,如果水体环境因子(如温度过高或氧含量过低等)不适时种群开始进行有性生殖产生休眠卵沉入水体沉积物中,保证种群的延续。研究表明,萼花臂尾轮虫群体是由隐蔽种复合体组成而且一般认为在野外水体中,在周期性孤雌生殖轮虫种群刚开始建立时有很高的基因型多样性,但在孤雌生殖阶段会受到克隆选择的影响,种群基因型多样性随着季节更迭逐渐降低。因为周期性孤雌生殖轮虫生活史的特点,野外种群和实验室种群都不可避免的存在近交衰退现象,而种群的近交回避策略又是这种独特交配系统的进化动力。然而,国际上对周期性孤雌生殖生物的基因型多样性和近交衰退现象的关注较少,国内也几乎没有开展过相关工作。本研究在国内外相关研究的基础上,以大连儿童公园明泽湖中萼花臂尾轮虫为研究对象,基于ITS(internal transcribed spacer,内部转录间隔区)和微卫星分子标记技术,探讨以下几个问题(1)小型人工水体是否存在隐蔽种复合体(2)在野外水体中萼花臂尾轮虫是否会经历克隆选择效应以及存在近交衰退现象(3)萼花臂尾轮虫微卫星位点突变率的高低。主要研究结果如下:1.本次研究以明泽湖中萼花臂尾轮虫实验种群为基础,通过转录组测序共获得1141个潜在的多态性微卫星位点,以萼花臂尾轮虫实验种群为对象,通过聚丙烯酰胺凝胶电泳,分析了其中150个微卫星位点的多态性水平,共获得单态性位点22个;两种多态性位点17个;三种多态性位点7个;四种多态性位点5个;五种多态性位点3个;同时结合已经公布的具有高多态性的萼花臂尾轮虫微卫星位点(12个),最后确定利用7个微卫星位点进行萼花臂尾轮虫野外种群遗传结构和微卫星位点突变率分析。2.为确定明泽湖中萼花臂尾轮虫隐蔽种复合体的组成,本次研究应用ITS分子标记技术,通过比较ITS基因序列差异(>4%为不同的隐蔽种)以及构建系统发育树,发现明泽湖中萼花臂尾轮虫隐蔽种复合体由Clade1和Clade2构成。与国际发表的萼花臂尾轮虫分类标准相结合,最终确定明泽湖中Clade1为隐蔽种D,拉丁名为B.calyciflorus s.s.;Clade2为隐蔽种C,拉丁名为B.fernandoi sp.nov.。为方便后续结果的描述,定义Clade1为BC-D;Clade2为BC-C。因为BC-D对温度的耐受性比BC-C低,所以BC-D在早春出现一段时间后被BC-C替代。3.本次研究利用7个微卫星分子标记分析了BC-D和BC-C的遗传多样性,结果表明BC-D群体的观测等位基因数和有效等位基因数分别为4.9286和2.8138;BC-C群体的观测等位基因数和有效等位基因数分别为3.4078和2.2552。在BC-D和BC-C群体中观测杂合度和期望杂合度分别为0.6077和0.6123以及0.6918和0.5203;Nei氏期望杂合度分别为0.5932和0.5123。表明野外萼花臂尾轮虫种群遗传多样性较高。但Hardy-Weinberg遗传平衡检验,每个种群至少有2个位点偏离Hardy-Weinberg平衡。遗传偏离指数D显示种群处在杂合子过剩状态。种群偏离Hardy-Weinberg平衡状态可能是杂合子过剩导致。2个稀有等位基因的频率变化反映出萼花臂尾轮虫中存在一定程度的近交衰退现象。通过基因型多样性分析表明萼花臂尾轮虫种群基因型多样性较高,而且在随着种群的演替经历了克隆选择效应。4.为了确定野外萼花臂尾轮虫种群杂合子过剩的原因,我们进行了萼花臂尾轮虫微卫星位点突变率的相关研究。本次研究检测实验室克隆培养的4个萼花臂尾轮虫种群约30和50世代在7个微卫星位点的数据,计算得到4个克隆种群的微卫星位点突变率。结果显示30世代时7个微卫星位点平均每个世代突变率在8.25×10~(-4)—3.33×10~(-2)之间,所有微卫星位点平均每个世代突变率为1.37×10~(-2);50世代时7个微卫星位点平均每个世代突变率在0—2×10~(-2)之间,所有微卫星位点平均每个世代突变率为4.65×10~(-3)。对比其他研究结果,萼花臂尾轮虫微卫星位点的突变率处在相对较高的水平。通过比较野外萼花臂尾轮虫种群和实验室4个克隆种群的等位基因频率分布可知捕食压力促进了稀有等位基因的产生。而且在遗传结构上均表现出杂合子过剩的状态,说明杂合子过剩是微卫星位点较高的突变率导致的。综上所述,大连儿童公园明泽湖中萼花臂尾轮虫种群是由非重叠的隐蔽种复合体组成,分别为隐蔽种BC-C(B.calyciflorus s.s.)和BC-D(B.fernandoi sp.nov)。两个种群均具有较高的遗传多样性。同时在温度和晶囊轮虫的双重压力下,导致BC-D在水体中消失,但由于休眠卵库的补充,孵化出BC-C继续种群的繁衍。BC-C的遗传结果表明种群经历了克隆选择效应并且种群内存在一定程度的近交衰退现象。在捕食压力下和微卫星位点突变率较高的情况下,萼花臂尾轮虫种群在倾向孤雌繁殖的同时有产生新的等位基因,使得杂合子过剩,导致种群偏离Hardy-Weinberg平衡。

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.

Study on the Genetic Diversity of the Field Population of Brachionus Calycifloruss

张吉

殷旭旺

硕士

大连海洋大学

萼花臂尾轮虫;;ITS;;微卫星;;遗传多样性;;突变率

周期性孤雌生殖的萼花臂尾轮虫(Brachionus calyciflorus)是淡水生态系统中浮游动物的重要种类,并且是群体遗传结构研究中常见的模式生物。生活史包括孤雌生殖和有性生殖两个阶段,种群刚建立是水体沉积物中大量休眠卵孵化出非混交雌体进行孤雌生殖快速扩增种群,如果水体环境因子(如温度过高或氧含量过低等)不适时种群开始进行有性生殖产生休眠卵沉入水体沉积物中,保证种群的延续。研究表明,萼花臂尾轮虫群体是由隐蔽种复合体组成而且一般认为在野外水体中,在周期性孤雌生殖轮虫种群刚开始建立时有很高的基因型多样性,但在孤雌生殖阶段会受到克隆选择的影响,种群基因型多样性随着季节更迭逐渐降低。因为周期性孤雌生殖轮虫生活史的特点,野外种群和实验室种群都不可避免的存在近交衰退现象,而种群的近交回避策略又是这种独特交配系统的进化动力。然而,国际上对周期性孤雌生殖生物的基因型多样性和近交衰退现象的关注较少,国内也几乎没有开展过相关工作。本研究在国内外相关研究的基础上,以大连儿童公园明泽湖中萼花臂尾轮虫为研究对象,基于ITS(internal transcribed spacer,内部转录间隔区)和微卫星分子标记技术,探讨以下几个问题(1)小型人工水体是否存在隐蔽种复合体(2)在野外水体中萼花臂尾轮虫是否会经历克隆选择效应以及存在近交衰退现象(3)萼花臂尾轮虫微卫星位点突变率的高低。主要研究结果如下:1.本次研究以明泽湖中萼花臂尾轮虫实验种群为基础,通过转录组测序共获得1141个潜在的多态性微卫星位点,以萼花臂尾轮虫实验种群为对象,通过聚丙烯酰胺凝胶电泳,分析了其中150个微卫星位点的多态性水平,共获得单态性位点22个;两种多态性位点17个;三种多态性位点7个;四种多态性位点5个;五种多态性位点3个;同时结合已经公布的具有高多态性的萼花臂尾轮虫微卫星位点(12个),最后确定利用7个微卫星位点进行萼花臂尾轮虫野外种群遗传结构和微卫星位点突变率分析。2.为确定明泽湖中萼花臂尾轮虫隐蔽种复合体的组成,本次研究应用ITS分子标记技术,通过比较ITS基因序列差异(>4%为不同的隐蔽种)以及构建系统发育树,发现明泽湖中萼花臂尾轮虫隐蔽种复合体由Clade1和Clade2构成。与国际发表的萼花臂尾轮虫分类标准相结合,最终确定明泽湖中Clade1为隐蔽种D,拉丁名为B.calyciflorus s.s.;Clade2为隐蔽种C,拉丁名为B.fernandoi sp.nov.。为方便后续结果的描述,定义Clade1为BC-D;Clade2为BC-C。因为BC-D对温度的耐受性比BC-C低,所以BC-D在早春出现一段时间后被BC-C替代。3.本次研究利用7个微卫星分子标记分析了BC-D和BC-C的遗传多样性,结果表明BC-D群体的观测等位基因数和有效等位基因数分别为4.9286和2.8138;BC-C群体的观测等位基因数和有效等位基因数分别为3.4078和2.2552。在BC-D和BC-C群体中观测杂合度和期望杂合度分别为0.6077和0.6123以及0.6918和0.5203;Nei氏期望杂合度分别为0.5932和0.5123。表明野外萼花臂尾轮虫种群遗传多样性较高。但Hardy-Weinberg遗传平衡检验,每个种群至少有2个位点偏离Hardy-Weinberg平衡。遗传偏离指数D显示种群处在杂合子过剩状态。种群偏离Hardy-Weinberg平衡状态可能是杂合子过剩导致。2个稀有等位基因的频率变化反映出萼花臂尾轮虫中存在一定程度的近交衰退现象。通过基因型多样性分析表明萼花臂尾轮虫种群基因型多样性较高,而且在随着种群的演替经历了克隆选择效应。4.为了确定野外萼花臂尾轮虫种群杂合子过剩的原因,我们进行了萼花臂尾轮虫微卫星位点突变率的相关研究。本次研究检测实验室克隆培养的4个萼花臂尾轮虫种群约30和50世代在7个微卫星位点的数据,计算得到4个克隆种群的微卫星位点突变率。结果显示30世代时7个微卫星位点平均每个世代突变率在8.25×10~(-4)—3.33×10~(-2)之间,所有微卫星位点平均每个世代突变率为1.37×10~(-2);50世代时7个微卫星位点平均每个世代突变率在0—2×10~(-2)之间,所有微卫星位点平均每个世代突变率为4.65×10~(-3)。对比其他研究结果,萼花臂尾轮虫微卫星位点的突变率处在相对较高的水平。通过比较野外萼花臂尾轮虫种群和实验室4个克隆种群的等位基因频率分布可知捕食压力促进了稀有等位基因的产生。而且在遗传结构上均表现出杂合子过剩的状态,说明杂合子过剩是微卫星位点较高的突变率导致的。综上所述,大连儿童公园明泽湖中萼花臂尾轮虫种群是由非重叠的隐蔽种复合体组成,分别为隐蔽种BC-C(B.calyciflorus s.s.)和BC-D(B.fernandoi sp.nov)。两个种群均具有较高的遗传多样性。同时在温度和晶囊轮虫的双重压力下,导致BC-D在水体中消失,但由于休眠卵库的补充,孵化出BC-C继续种群的繁衍。BC-C的遗传结果表明种群经历了克隆选择效应并且种群内存在一定程度的近交衰退现象。在捕食压力下和微卫星位点突变率较高的情况下,萼花臂尾轮虫种群在倾向孤雌繁殖的同时有产生新的等位基因,使得杂合子过剩,导致种群偏离Hardy-Weinberg平衡。

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.