Understanding the origin of parthenogenesis

了解孤雌生殖的起源

• 批准号: 10188567
• 负责人: Sen Xu
• 金额: $ 37.94万
• 依托单位: UNIVERSITY OF TEXAS ARLINGTON
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-08-01 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10188567
• 关键词: Alleles; Animals; Backcrossings; Biological Assay; Biology; CRISPR/Cas technology; Candidate Disease Gene; Cytology; Daphnia; Diploidy; Environment; Fresh Water; Gene Expression; Genes; Genetic; Genome; Genomic approach; Germ Cells; Goals; Knowledge; Life Cycle Stages; Mediating; Meiosis; Modification; Natural History; Oocytes; Outcome; Ovary; Parthenogenesis; Partner in relationship; Periodicity; Phenotype; Play; Population; Public Health; RNA Interference; Regulation; Regulator Genes; Reproduction; Role; Sexual Reproduction; System; Testing; Tissue-Specific Gene Expression; asexual; base; differential expression; experience; functional genomics; genomic tools; innovation; insight; interest; knock-down; novel; reproductive; response; screening; sex; transcriptome sequencing

Project Summary Parthenogenesis (i.e., reproduction without mating) has evolved from sexual reproduction in nearly all major eukaryotic groups. In parthenogenesis, chromosomally unreduced (e.g., diploid) gametes result from modified forms of meiosis. Understanding the genetic mechanisms underlying the modification of meiosis in parthenogenetic lineages is of significant public health interest because meiosis is central to sexual reproduction. Using parthenogenesis to understand the genetic regulation of meiosis is also a highly innovative approach, with its natural history perspective most likely yielding novel knowledge about meiosis. Using a combination of evolutionary and functional genomic approaches, this project examines the genetic bases of cyclical and obligate parthenogenesis in the freshwater microcrustacean Daphnia, which represents an excellent tractable experimental system with well-understood biology and many genomic tools. Daphnia is well known for its cyclical parthenogenesis (CP) life cycle, i.e., propagating asexually under favorable environmental conditions and switching to sexual reproduction in response to stressful environment. Interestingly, some populations of the species D. pulex (backcrosses of two parental CP species D. pulex and D. pulicaria) reproduce by obligate parthenogenesis (OP) because they lost the capability to engage in sex. This project has two long-term goals. First, considering that a single Daphnia genome can encode the genetic machinery for both reproductive strategies, it is hypothesized that environment-mediated gene expression changes, especially the neofunctionalization of duplicated meiosis genes (e.g. Cdc20), play a role in the origin of CP. To test this, ovary- specific gene expression changes between parthenogenesis and meiosis in CP D. pulex and D. pulicaria will be examined to produce a high quality set of candidate genes. To determine the functional role of these candidates in CP (e.g., master regulator genes), we will perform RNAi knockdown of each candidate in CP isolates and examine the associated phenotypic effects using animal reproduction assay and cytological examination of developing oocytes. Second, concerning the origin of OP, we hypothesize that the genetic incompatibility between the two ecologically divergent species CP D. pulex and D. pulicaria is a key factor. For the previously identified candidate genes for OP, we will test (1) whether they experience differential gene expression in OP isolates compared to CP isolates and (2) whether their expression is mis-regulated (i.e., genetic incompatibility). The former will be achieved by ovary-specific RNA-seq in OP and CP clones at different reproductive stages, whereas the latter will be based on allele-specific RNA-seq analysis of the parental species and OP isolates. For the candidate genes showing differential expression and mis-regulation, RNAi knockdown and phenotype screening will be performed to verify their functional role in OP, whereas CRISPR/Cas9 editing will be used to identify the causal SNPs for each gene. Lastly, we will re-create OP animals by introducing OP-causal SNPs to CP Daphnia pulex using CRISPR/Cas9 editing.

项目摘要 单性生殖（即，没有交配的繁殖）几乎在所有主要的物种中都从有性繁殖进化而来。 真核生物群在单性生殖中，染色体未还原（例如，二倍体）配子由修饰的 减数分裂的形式。了解减数分裂修饰的遗传机制， 单性生殖谱系具有重大的公共卫生意义，因为减数分裂是有性生殖的核心。 利用孤雌生殖来了解减数分裂的遗传调控也是一种高度创新的方法， 它的自然历史观点很可能产生关于减数分裂的新知识。结合使用 进化和功能基因组学方法，该项目研究了周期性和专性的遗传基础， 孤雌生殖在淡水微甲壳类水蚤，这是一个很好的听话 实验系统与充分理解的生物学和许多基因组工具。水蚤以其周期性而闻名 孤雌生殖（CP）生命周期，即，在有利的环境条件下无性繁殖， 为了应对压力环境而转向有性生殖。有趣的是， 种D. pulex（两个亲本CP种D. pulex和D.通过专性繁殖 孤雌生殖（OP），因为他们失去了参与性行为的能力。该项目有两个长期目标。 首先，考虑到一个单一的水蚤基因组可以编码的遗传机制，为生殖 策略，假设环境介导的基因表达变化，特别是 重复的减数分裂基因（例如Cdc 20）的新功能化在CP的起源中起作用。为了验证这个，卵巢- CP D孤雌生殖与减数分裂之间特异基因表达的变化。pulex和D. pulicaria将是 以产生一组高质量的候选基因。确定这些候选人的职能作用 在CP中（例如，主调节基因），我们将在CP分离株中对每种候选物进行RNAi敲低， 使用动物繁殖试验和细胞学检查检查相关的表型效应， 发育中的卵母细胞其次，关于OP的起源，我们假设遗传不相容性 两个生态上不同的物种CP D之间的关系。pulex和D.蚤类是一个关键因素。的先前 确定了OP的候选基因，我们将测试（1）它们是否在OP中经历差异基因表达 分离株与CP分离株相比的差异和（2）它们的表达是否被错误调节（即，遗传不相容性）。 前者将在不同生殖阶段的OP和CP克隆中通过卵巢特异性RNA-seq实现， 而后者将基于亲本物种和OP分离株的等位基因特异性RNA-seq分析。为 差异表达和误调节的候选基因、RNAi敲除和表型 将进行筛选以验证它们在OP中的功能作用，而CRISPR/Cas9编辑将用于 确定每个基因的致病SNP。最后，我们将通过引入OP-因果SNP来重建OP动物， 使用CRISPR/Cas9编辑的CP蚤状水蚤。