A robust Bayesian two-sample test for detecting intervals of differential gene expression in microarray time series.

Understanding the regulatory mechanisms that are responsible for an organism's response to environmental change is an important issue in molecular biology. A first and important step towards this goal is to detect genes whose expression levels are affected by altered external conditions. A range of methods to test for differential gene expression, both in static as well as in time-course experiments, have been proposed. While these tests answer the question whether a gene is differentially expressed, they do not explicitly address the question when a gene is differentially expressed, although this information may provide insights into the course and causal structure of regulatory programs. In this article, we propose a two-sample test for identifying intervals of differential gene expression in microarray time series. Our approach is based on Gaussian process regression, can deal with arbitrary numbers of replicates, and is robust with respect to outliers. We apply our algorithm to study the response of Arabidopsis thaliana genes to an infection by a fungal pathogen using a microarray time series dataset covering 30,336 gene probes at 24 observed time points. In classification experiments, our test compares favorably with existing methods and provides additional insights into time-dependent differential expression.

Saline-saturated DMSO-EDTA as a storage medium for microbial DNA analysis from coral mucus swab samples

The mucus surface layer of corals plays a number of integral roles in their overall health and fitness. This mucopolysaccharide coating serves as vehicle to capture food, a protective barrier against physical invasions and trauma, and serves as a medium to host a community of microorganisms distinct from the surrounding seawater. In healthy corals the associated microbial communities are known to provide antibiotics that contribute to the coral’s innate immunity and function metabolic activities such as biogeochemical cycling. Culture-dependent (Ducklow and Mitchell, 1979; Ritchie, 2006) and culture-independent methods (Rohwer, et al., 2001; Rohwer et al., 2002; Sekar et al., 2006; Hansson et al., 2009; Kellogg et al., 2009) have shown that coral mucus-associated microbial communities can change with changes in the environment and health condition of the coral. These changes may suggest that changes in the microbial associates not only reflect health status but also may assist corals in acclimating to changing environmental conditions. With the increasing availability of molecular biology tools, culture-independent methods are being used more frequently for evaluating the health of the animal host. Although culture-independent methods are able to provide more in-depth insights into the constituents of the coral surface mucus layer’s microbial community, their reliability and reproducibility rely on the initial sample collection maintaining sample integrity. In general, a sample of mucus is collected from a coral colony, either by sterile syringe or swab method (Woodley, et al., 2008), and immediately placed in a cryovial. In the case of a syringe sample, the mucus is decanted into the cryovial and the sealed tube is immediately flash-frozen in a liquid nitrogen vapor shipper (a.k.a., dry shipper). Swabs with mucus are placed in a cryovial, and the end of the swab is broken off before sealing and placing the vial in the dry shipper. The samples are then sent to a laboratory for analysis. After the initial collection and preservation of the sample, the duration of the sample voyage to a recipient laboratory is often another critical part of the sampling process, as unanticipated delays may exceed the length of time a dry shipper can remain cold, or mishandling of the shipper can cause it to exhaust prematurely. In remote areas, service by international shipping companies may be non-existent, which requires the use of an alternative preservation medium. Other methods for preserving environmental samples for microbial DNA analysis include drying on various matrices (DNA cards, swabs), or placing samples in liquid preservatives (e.g., chloroform/phenol/isoamyl alcohol, TRIzol reagent, ethanol). These methodologies eliminate the need for cold storage, however, they add expense and permitting requirements for hazardous liquid components, and the retrieval of intact microbial DNA often can be inconsistent (Dawson, et al., 1998; Rissanen et al., 2010). A method to preserve coral mucus samples without cold storage or use of hazardous solvents, while maintaining microbial DNA integrity, would be an invaluable tool for coral biologists, especially those in remote areas. Saline-saturated dimethylsulfoxide-ethylenediaminetetraacetic acid (20% DMSO-0.25M EDTA, pH 8.0), or SSDE, is a solution that has been reported to be a means of storing tissue of marine invertebrates at ambient temperatures without significant loss of nucleic acid integrity (Dawson et al., 1998, Concepcion et al., 2007). While this methodology would be a facile and inexpensive way to transport coral tissue samples, it is unclear whether the coral microbiota DNA would be adversely affected by this storage medium either by degradation of the DNA, or a bias in the DNA recovered during the extraction process created by variations in extraction efficiencies among the various community members. Tests to determine the efficacy of SSDE as an ambient temperature storage medium for coral mucus samples are presented here.

采用AFLP分子标记和分子生物学方法对资源植物的评价

中国资源植物丰富，蕴藏着优异的基因资源，开发和利用这些优异资源是植物学研究的重要课题。本文面向国家重大需求选择两种资源植物一羊草(Leymuschinensis (Trin.)Tzvel)和向日葵(llelia thus annuus L．)，采用分子标记技术和分子生物学方法对其进行评价和研究，以期为资源利用提供依据。由于两种植物本身的差别和采用的研究方法各异，故分别论述。 羊草，隶属禾本科赖草属，是欧亚大陆草原区东部重要建群种之一。羊草是牧草之王，是我国比较有优势的战略性生物资源，对我国北方畜牧业的发展以及生态环境的保育均具有重要意义。近年来，由于缺乏科学管理、过度放牧等不利影响，加之羊草本身固有的“三低”问题（即抽穗率低、结实率低、发芽率低）已对羊草生物多样性维持构成了严重的威胁，限制了我国人工草地建设和天然草地的改良及沙化治理的步伐。因此，如何通过形态调查结合生物技术手段评价羊草遗传多样性为建立核心种质及改良羊草、快速评价和创造新的种质、如何加快育种进程便成为当前亟待解决的问题。本文围绕这些问题开展了系统的研究并取得如下结果： 1． 对羊草的形态调查和AFLP分析，表明羊草是一种形态变异较大但是遗传变 异较小的物种。两种生态型的表现显著差异，其中灰绿生态型羊草比黄绿生 态型差异大。羊草遗传多样性与包括长期的栽培驯化、地理分布有很大的相 关性，地理来源相同的几乎全部聚到了一组。 2． 通过主成分分析和通径分析，简化了羊草31个性状分析的复杂性，了解到 羊草无性繁殖受好的营养生长促进。 3． AFLP分子标记技术在分析羊草遗传多样性方面有显著优势，尤其是对于羊 草这样多态性不高的物种是一种非常有效的分析工具。在分析AFLP数据时 采用聚类分析和主坐标分析相结合的方法，既兼顾了亲缘关系较近的种质之 间的关系调查也兼顾了亲缘关系较远的种质之间的关系调查。 4．羊草AFLP反应，不同引物所获得的总带数和多态性带数差别明显。羊草基因 组对3’端有选择性碱基TN的所有EcoRJ选择性引物扩增效果很差，前人 的有关赖草属的遗传研究也支持这一结果。 向日葵(n=17)，属于菊科( Compositae)向日葵属(Helia thus)，向日葵的研究重要领域是向日葵杂种生产，而细胞质雄性不育系的使用是杂种优势育种的核心。全世界90%以上的向日葵杂交种生产仍然在使用同一个细胞质类型PETI，玉米遗传单一给生产带来的毁灭性打击仍然令研究者和生产者记忆犹新，因此寻找更多的细胞质类型仍然是研究者的重要任务。本研究围绕一个新的不育源(G20023)的发现及鉴定，通过使用不育的G20023的保持系、恢复系、恢复的Fi代、回交一代之间比较以及与属于PETI细胞质类型的不育系的相应材料进行比较，找出与这一新的细胞质类型不育表型有关的可能差异序列，来探讨其不育机制，得到如下结果： 1、 通过田间杂交试验，证明G20023的保持系有很多（已证实有24份）， 目前找到的恢复系只有一个，H.maximiliani。G20023不育源作为一 种新的细胞质类型可以成为将来杂交育种的候选资源。同时，我们找 到一些表型证据，除了无花粉之外，G20023与PETI表型的典型不同 之处还在于前者的花药上下均为分离状态，而后者花药的基部联合， 顶部分离。显然，不同的细胞质类型在解剖结构上可能表现不尽相同。 2、 与线粒体基因组特异基因的核酸序列比较，结果表明，G20023线粒体 基因组上没有orfH522序列，与PETI表现出差异;此外，在基因atp6 位点也与PETI不同，而且在该位点也与同属向日葵ANTI不相同。同 时由于orf873并没有出现在ANTI中而出现在G20023中，因此我们可 以认为G20023这一个新的不育系是与ANTI和PETI不同的细胞质类 型。 3、 在参考常规线粒体DNA提取方法的基础上，我们做了很多改进，建立 了自己的向日葵线粒体DNA提取方法。该方法更快更简单，提取的线 粒体DNA完全可以用于酶切和杂交。 4、 G20023不育源由于其稳定的不育性状，可以作为培育无花粉彩色向日 葵杂交种的亲本材料，我们通过此不育源选育适当花色的无花粉观赏 向日葵生产杂交种。