DNA-methylation to improve conservation of TSD species

全球变暖威胁着400多种具有温度依赖性性别决定(TSD)的物种，包括所有海龟。在佛得角--世界上最大的龟类聚集地--预计增加3摄氏度将导致极端的性别比偏差，到2100年将有99%的女婴出生。如果这些预测是正确的，这个种群和物种很快就会灭绝。为了减少这种可能性，我们需要对性别决定有更好的分子理解，并将其应用于新的管理策略。自从海龟在大约1.2亿年前首次出现以来，它们一直暴露在剧烈的气候变化中。因此，它们很可能进化出了尚不为人所知的分子机制，缓冲了温度变化导致的极端性别比偏见。然而，这些机制可能跟不上当前全球变暖的步伐，因为我们已经看到成年女性占95%的人口。但相比之下，如果他们这样做了，我们预测未来的模型是错误的。因此，我们可能需要重新考虑缓解策略，比如孵化室，这些策略最大限度地提高了新生儿的存活率，但产生了未知的性别比例，甚至可能放大了这个问题。在海龟中，性别决定是一种可塑性特征，只有关键温度的微小变化才能使整个海龟窝由雄性或雌性组成。从机械论的角度来看，表观遗传过程经常被提出来控制表型可塑性，这可以加速对自然选择的适应性反应。虽然有几种表观遗传机制可以促进表型可塑性，但DNA甲基化--在胞嘧啶核苷酸上加一个甲基--是迄今为止最具特性的机制之一。DNA甲基化特别有趣，因为我们可以区分环境诱导的标记和可能的遗传标记。虽然，了解这种机制是否存在具有全球保护重要性，但在此之前，我们需要创新方法，使新生儿能够在不像目前情况下杀死他们的情况下进行性别鉴定。在这里，我们将利用作为申请者领导的NERC项目(NE/V001469/1)的一部分进行的大型现场实验。具体地说，我们收集了筑巢的斑海龟的血液样本，并将它们的卵窝移到一个实验孵化室，在那里它们暴露在自然孵化条件下，但受到保护，不会被捕食。每个卵被分成两组，分别暴露在雄性孵化温度(深层)或雌性孵化温度(浅层)中，以模拟未来的气候变暖。孵化后，我们采集血液样本并进行体能测试，以确定温度对新生儿散布的影响。鉴于这项实验的成功完成，我们提出对新生儿和母亲的血液样本进行全基因组亚硫酸氢盐测序(WGBS)，以确定全基因组DNA甲基化标记。在我们新注释的高质量参考基因组的支持下，我们将识别与出生时性别决定有关的基因和基因网络，并将黄金标准WGBS与便携式测序解决方案(如Minion)进行比较。至关重要的是，因为这个项目的重点是提供基于保护的解决方案，所以我们没有调查早期的发展阶段，因为它们需要牺牲个体，因此不太可能阐明应对气候变暖挑战所需的保护策略。总体而言，这个项目将填补保护方面的主要空白，通过确定海龟在全球变暖面前的潜在持久性，并提供实用的诊断工具来非致命性地确定出生时的产卵性别比例；ii)在进化生物学方面，因为它将提供与温度性别决定相关的机制的新理解。虽然这个项目的重点是海龟，但它的意义延伸到了患有TSD的400种脊椎动物。

Global warming threatens over 400 species that have temperature-dependent sex determination (TSD), including all sea turtles. In Cabo Verde - home of the world's largest aggregation of loggerhead turtles (Caretta caretta) - a predicted increase of 3C will result in an extreme sex ratio bias, with >99% female neonates by 2100. If these predictions are correct, this population, and species, will go extinct soon. To mitigate that possibility, we need a better molecular understanding of sex determination that can be applied to new management strategies.Since they first appeared, ~120 million years ago, sea turtles have been exposed to dramatic climatic changes. It is therefore likely they have evolved still undescribed molecular mechanisms that buffer against extreme sex ratio bias driven by temperature variation. Yet, these mechanisms may not keep up with the pace of current global warming, as we already see populations with >95% adult females3. But in contrast, if they do, our models that predict the future are wrong. Hence we may need rethinking mitigation strategies such as hatcheries that maximise the survival of neonates but produce unknown sex ratios, and may even amplify the problem.In sea turtles, sex determination is a plastic trait with only a narrow variation in the pivotal temperature making an entire clutch being composed of either males or females. From a mechanistic perspective, epigenetic processes have regularly been proposed to control phenotypic plasticity that can accelerate adaptive responses to natural selection. Although several epigenetic mechanisms can facilitate phenotypic plasticity, DNA methylation - the addition of a methyl group to cytosine nucleotides - is one of the best characterized to date. DNA methylation is particularly interesting because, we can differentiate environmentally-induced marks and possibly inherited ones. While, understanding if such mechanisms exist is of global conservation importance, before that we need to innovate methods that enable sexing neonates without killing them as is currently the case. Here, we will take advantage of a large field experiment conducted as part of a NERC project led by the applicant (NE/V001469/1). Specifically, we collected blood samples of nesting loggerhead turtles, and moved their egg clutches into an experimental hatchery, where they were exposed to natural incubation conditions, but were protected from predation. Each egg clutch was split into two groups to be exposed to male incubating temperature (deeper depth) or female incubating temperature (shallow depth), mimicking future climate warming. Upon hatching, we collected blood samples and conducted fitness tests to determine the effect of temperatures on dispersal of neonates. Given the successful completion of this experiment, we offer to sequence neonates and mother blood samples for whole-genome bisulfite sequencing (WGBS) to determine genome-wide DNA methylation marks. Backed up by our newly annotated high quality reference genome, we will identify the genes and gene networks involved in sex determination at birth and compare the gold standard WGBS with portable sequencing solution such as MinION. Crucially, because this project focuses on offering conservation-based solutions, we do not investigate the early development stages, since they would require sacrificing individuals and are therefore unlikely to shed light onto conservation strategies needed to respond to the challenge of climate warming.Overall, this project will fill major gaps i) in conservation by determining the potential persistence of sea turtles in the face of global warming and offering practical diagnostic tools to non-lethally determine a clutch sex ratio at birth; ii) in evolutionary biology, because it will provide a new understanding of the mechanisms associated with temperature sex determination. While this project focuses on sea turtles, its significance extends to >400 vertebrate species with TSD.