Molecular dissection of paramutation in tomato

许多特征是根据众所周知的孟德尔法则遗传的：棕色眼睛的父亲和蓝色眼睛的母亲将有50%或100%的棕色眼睛孩子，这取决于父亲是从父母中的一方还是双方那里遗传了显性的棕色眼睛基因。反过来，他们的棕色眼睛的孩子与蓝眼睛的伴侣将产生50%的棕色眼睛后代。这种情况下的蓝眼睛是遗传隐性的，因为该基因没有功能--编码的蛋白质缺乏产生眼睛色素的能力。在大多数情况下，植物遗传学同样简单明了，每个基因都有显性和隐性版本。然而，也有一些例外，相当于蓝眼睛父母的所有第一代和后续后代都有蓝色的眼睛，即使另一位父母的眼睛是棕色的。在这种情况下，基本的机制被称为参数转换。这项建议旨在了解辅助突变是如何发挥作用的，这样我们就可以在未来评估其在自然种群进化中的重要性以及在农作物中应用的潜力。我们所知道的关于辅助突变的大部分涉及植物基因，就像人类的眼睛颜色基因一样，编码色素决定因素。然而，这些系统中的沉默基因不同于蓝眼基因，因为它们不是遗传隐性的。它们是显性的，因为在第一代后代(F1)中，来自非有色亲本的基因的沉默状态被转移到来自有色亲本的同等基因。这种后代的两种色素基因都是沉默的。然而，情况比正常的显性和隐性更复杂，因为以前活跃的基因已经永久性地改变了。它的沉默状态现在遗传给后代，就像最初来自色素减去亲本的基因一样，它可以在后续世代将其沉默状态转移到活跃版本的基因上。一种可能的解释是，沉默的基因具有某种突变性，可以改变活性基因的序列。然而，从早期的研究中，我们知道在玉米和番茄中，活跃基因和沉默基因的DNA序列是相同的。新出现的图景涉及影响基因表达的DNA和相关染色体蛋白的化学修饰--甲基化。染色质结构和RNA也有牵连。我们将这些因素称为表观遗传而不是遗传，因为DNA序列没有变化。然而，以前的研究没有显示沉默的表观遗传标记是如何转移到活跃版本的基因上的：这两个基因是否存在直接接触，或者沉默的基因是否产生了一种扩散因子--也许是一种RNA--可以与活跃的基因结合？同样，我们也不明白为什么一些与辅助突变过程中的基因具有相同化学修饰的基因不是副突变基因。这些其他基因可能是沉默的，它们的沉默可能是可遗传的，但沉默状态是以正常的孟德尔模式遗传的。为了解决这些问题，我们提议使用西红柿，我们最近在西红柿中确定了一个辅助切割目标。该体系中色素的损失导致了壮观的黄色黄化--硫脲。我们建议利用基因编辑技术敲除可能影响硫脲参数化的基因，并删除硫脲基因座上的DNA，以确定它是否影响顺突变或可参数突变。番茄作为解决这些问题的实验系统有许多优点--它适用于针对DNA甲基化的病毒基因沉默系统-VIGS，我们最近的工作发现了其他可以与硫脲进行比较和对比的辅助突变基因座。

Many traits are inherited according to the well known Mendelian rules: a brown-eyed father and blue-eyed mother will have either 50% or 100% brown eyed children depending on whether the father inherited dominant brown eye genes from one or both of his parents. In turn, their brown-eyed children with a blue-eyed partner would produce 50% brown eyed offspring. Blue eyes in this situation is genetically recessive because the gene is non functional - the encoded protein lacks the ability to produce eye pigments. Plant genetics, in most instances, is similarly straightforward with each gene having dominant and recessive versions. There are however are some exceptions that are the equivalent of a situation in which all first and subsequent progeny of a blue eyed parent have blue eyes even if the other parent has brown eyes. In this situation the underlying mechanism is referred to as paramutation. This proposal aims to understand how paramutation works so that, in future, we can assess its importance in the evolution of natural populations and the potential for its application in crops.Much of what we know about paramutation involves plant genes that, like eye colour genes in people, encode pigment determinants. The silent genes in these systems are, however, unlike the blue-eye genes in that they are not genetically recessive. They are dominant because, in the first generation offspring (F1) the silent state of the gene from a non-pigmented parent is transferred to the equivalent gene from the pigmented parent. Both pigment genes in this offspring are silent. The situation is, however, more complicated than normal dominance and recessiveness because the previously active gene has been permanently changed. Its silent state is now inherited into subsequent generations and, like the original gene from the pigment minus parent, it can transfer its silent state in subsequent generations to an active version of the gene. A possible explanation of paramutation is that the silent gene is somehow mutagenic and can change the sequence of the active. From earlier studies, however, in maize and tomato we know that the DNA sequence of the active and silent genes is identical. The emerging picture involves a chemical modification -methylation - of the DNA and of associated chromosomal proteins that affect gene expression. Chromatin structure and RNA has also been implicated. We refer to these factors as being epigenetic rather than genetic because there is no change to the DNA sequence. The previous studies of paramutation do not, however, show how the silencing epigenetic mark is transferred to the active version of the gene: is there direct contact of the two genes or does the silent gene produce a diffusible factor - an RNA perhaps - that can bind to the active gene? Similarly we do not understand why some genes with the same chemical modification as those in paramutation are not paramutagenic. These other genes may be silenced and their silencing may be heritable but the silenced state is inherited in a normal Mendelian pattern. To address these questions we are proposing to use tomato in which we have recently identified a target of paramutation. The loss of pigment in this system results in a spectacular yellow chlorosis - sulfurea. We propose to exploit gene editing technology to knock out genes that might influence sulfurea paramutation and we will delete DNA at the sulfurea locus to find out whether it affects either paramutagenicity or paramutability. Tomato has many advantages as an experimental system for these questions - it is amenable to a viral gene silencing system - VIGS - that can target DNA methylation and our recent work has identified other paramutation loci that can be compared and contrasted with sulfurea.