For a master internship (several months), we propose several projects around the study of Mesorhabditis belari, and in the continuity of our first results:
“Why would parthenogenetic females systematically produce males who do not transmit their genes to females?” [1] M. Grosmaire, C. Launay, M. Siegwald, M-A. Félix, P-H. Gouyon, M. Delattre. Under review in Science.
Project I. What controls the amphimixic versus gynogenetic fate of embryos ?
To answer to this question, we have already isolated the embryos of each category and sequenced their RNAs. We have identified reads that are specific of each category by comparative transcriptomic. To go further and understand the roles of these genes we wil i) confirm their differential expression by performing qRT-PCR on each category of embryos, ii) localize the ARNs in embryos and in animals with in situ hybridization, iii) analyse the spatio-temporal distribution of the corresponding proteins (when RNAs are coding sequences) using antibody stainings.
Project II. Is there a sexual chromosome in Mesorhabditis belari ?
Amphimixic embryos produce only males. In the case of a XX/XY system, we expected the production of 50% males and 50% females. Thus, sex determination could be non chromosomal, or if there is a sex chromosome, its repartition in sperm cells has to be highly biased. Because the cytological analysis of chromosomes in this species did not reveal any chromosome of a different shape, we performed a DNA sequencing of males and females separatately to look for sex specific reads. To our surprise, we found reads that are specific to males and other that are present in both sexes but highly duplicated in males, strongly suggesting that a Y chromosome is present in males. We will i) perform DNA Fish experiements to confirm that all these reads are present on the same chromosome (Y), ii) perform qRT-PCR on different male tissu to test whether the expression pattern of these genes is consistent with a role in the sex determination system, iii) identify homologs in the closest nematode species, that present a classical male/femelle reproductive system (to know if the common ancestor had a Y chromosome as well or if the system has diverged)
Project III. Are M. belari females clones of their mother ?
We want to know if the genome of M. belari females is stable (because of clonal expansion) or if it remains dynamic. After fertilization, when the male DNA it not utilized to produce gynogenetic embryos, the female meiosis in the oocyte is incomplete, which restores the ploidie in females. Depending on the type of meiotic division (being reductional, equational, with or without recombination, etc.), the genotype of daugthers might be identical or different from mothers. To analyze this, we will i) use bioinformatic approaches to establish the level of heterozygosity in females and to detect events of recombination, gene conversion etc. ii) detect heterozygote SNP and will analyze their distribution in the progeny of females (using PCR genotyping on a small scale or Bar-seq for a larger map of the genome), iii) analyze the figures of meiosis using immuno-histology to detect the number of crossing overs, DNA double strand breaks and to ultimately know with step of meiosis is skipped during the production of females.