蔷薇类的分子系统学研究—兼论系统发育基因组学的原理和方法(以SET基因超家族为例)

分子系统发育分析的主要任务包括：（1）帮助建立生命之树（tree of life）;（2）追踪基因和基因家族（gene family）的起源和进化, 以获知基因在进化过程中的功能分化和伴随发生的重要分子事件（key molecular events）和形态性状的关键创新（key innovation）。这两个方面在本研究中都有所涉及。对于前者，选用植物线粒体matR基因重建被子植物蔷薇类群的系统发育关系;对于后者，则以SET基因超家族为例，探讨其在真核生物中的进化分类以及与功能多样性的关系。 I 蔷薇类的分子系统学 蔷薇类（rosids）是基于分子数据建立的被子植物的主要分支之一，包含13个目，大约三分之一的被子植物物种。两个主要蔷薇类内部分支是豆类fabids（包含7个目）和锦葵类malvids（包含3个目）。然而，这两个分支内部，以及这两个分支与蔷薇类基部类群，包括牻牛儿苗目（Geraniales）、桃金娘目（Myrtales）和流苏子目（Crossosomatales）之间的关系大多是不清楚的。本研究中，我们选取174个物种来代表72个蔷薇类（rosids）的科，利用两个数据集，即线粒体matR单基因数据集和包括线粒体matR基因、两个质体基因(rbcL、 atpB)和一个核基因(18S rDNA) 的4基因数据集，重建蔷薇类在科以上分类阶元水平的系统发育关系。同时，还对线粒体matR基因的进化特征和用于大尺度系统发育分析的适合度和潜力进行了评价。 线粒体matR单基因数据支持malvids和大多数蔷薇类目的单系性质，然而，豆类（fabids）成员没有形成一个分支，其COM亚支，包括卫矛目（Celastrales）、酢浆草目（Oxalidales）、金虎尾目（Malpighiales）和蒜树科（Huaceae），分辨为锦葵类（malvids）的姐妹群。这个关系在最近根据花结构特征曾被提出过，但从未在之前的分子系统发育分析中得到分辨。4基因数据集支持首先是牻牛儿苗目（Geraniales），接着是桃金娘目（Myrtales）作为蔷薇类（rosids）的最基部的分支;流苏子目（Crossosomatales）是锦葵类（malvids）姐妹群，以及蔷薇类（rosids）的核心部分包括豆类（fabids），锦葵类（malvids）和流苏子目（Crossosomatales）。线粒体matR基因的进化特征分析显示，与两个叶绿体基因(rbcL 和atpB)比较，同义替代速率约是它们的1/4，而非同义替代速率接近于自身的同义替代速率，表明matR 基因具有松弛的选择压力。线粒体matR基因相对慢速的进化使非同源相似（homoplasious）突变减少，提高了系统发育信息的质量，同时，松弛的选择压力使非同义替代数量增加，弥补了慢速进化导致的系统发育信息数量不足的缺陷，这两个方面的结合使线粒体matR基因非常适用于被子植物在科以上水平的系统发育研究。 II SET基因超家族的系统发育基因组学分析 SET基因超家族基因编码含有SET结构域的蛋白，在真核生物中，SET-domain蛋白一般是多结构域（multi-domain）的。SET-domain蛋白具有对组蛋白H3和H4的N末端尾部进行赖氨酸残基甲基化修饰的酶活性;从异染色质形成到基因转录，甲基化的组蛋白广泛影响染色质水平的基因调控。依据SET结构域一级序列的相似性和结构域组织（domain architecture）特征，目前，SET-domain基因超家族被划分为4-7个家族。由于这些划分或者使用动物或者使用植物SET基因，只有少数其它类群的物种加入分析，因此这样的划分可能是不完整的。本研究采用系统发育基 因组学方法（phylogenomic approach），在真核生物范围内广泛取样，期望获得相对完整的SET-domain基因家族的 进化分类方案，在此基础上加深理解SET-domain基因的进化机制和功能多样性。 在提取了17个物种，代表5个真核超群的SET蛋白序列基础上，系统发育分析结合“结构域组织特征”鉴别了9个SET基因家族，其中一个是新的SET基因家族。以前的SET8和Class VI家族,及SMYD和SUV4-20家族分别合并为一个家族。大部分家族在进化过程中发生了2次以上的基因重复事件，通过获得不同的结构域产生具有不同功能的新基因。一个SET基因家族在进化过程中推测发生了从脊椎动物祖先向盘基网柄菌（Dictyostelium discoideum）的水平基因转移。

Discovery of a male-biased mutant family and identification of a male-specific SCAR marker in gynogenetic gibel carp Carassius auratus gibelio

Gibel carp ( Carassius auratus gibelio) is a uniquely gynogenetic species with a minor ratio of males in natural habitats, but its male origin and sex determination mechanisms have been unknown. In this study, a male-biased mutant family was discovered from the gynogenetic gibel carp, and a male-specific SCAR marker was identified from the mutant family. Normal spermatogenesis was observed in the male testes by immuno. fluorescence histochemistry. Nearly identical AFLP profiles were observed between males and females, but a male-specific 86 bp AFLP fragment was screened by sex-pool bulked segregant analysis and individual screening. Based on the male-specific AFLP fragment, a total of 579 bp sequences were cloned by genome walking. Subsequently, a male-specific SCAR marker was designed, and the male-specific DNA fragment was confirmed to be steadily transmitted to the next generation and consistently detected only in males. (C) 2009 National Natural Science Foundation of China and Chinese Academy of Sciences. Published by Elsevier Limited and Science in China Press. All rights reserved.

Microsatellite marker isolation and cultured strain identification in Carassius auratus gibelio

Microsatellites have become the preferred molecular markers for strain selection and genetic breeding in fish. In this study a total of 105 microsatellites were isolated and identified in gibel carp (Carassius auratus gibelio) by microsatellite sequence searches in GenBank and other databases and by screening and sequencing of positive clones from the genomic library enriched for AG and GATA repeats. Moreover, nineteen microsatellites were randomly selected to design locus-specific primer pairs, and these were successfully used to identify and discriminate different cultured strains of gibel carp including strains A, D, L, and F. Three different types of microsatellite pattern were distinguished by the number and length of fragments amplified from the 19 primer pairs, and some microsatellite primer pairs were found to produce different microsatellite patterns among strains and strain-specific fragments. In addition, some duplicated alleles were also detected in two microsatellite patterns. Therefore, the current study provides direct molecular markers to discriminate among different cultured strains for selective breeding and aquaculture practice of gibel carp.

Characterization of a highly conserved microsatellite marker with utility potentials in cyprinid fishes

A microsatellite locus, MFW1, originating from common carp is highly conserved in flanking nucleotides but variable in repeat length in some fishes from different families of the Cypriniformes. This orthologous locus is polymorphic in approximately 58% of the species tested in the order and is inherited by Mendelian law. It proved to be a potentially good marker in population genetics and in the cyprinid species-breeding programme in which no microsatellite markers were available.

Genetic structure of Chinese sucker population Myxocyprinus asiaticus in the Yangtze River based on mitochondrial DNA marker

The sequencing analysis of the mitochondrial DNA control region (mtCR DNA) was performed to assess the genetic divergence and population structure of the Chinese sucker Myxocyprinus asiaticus (Cypriniformes Catostomidae) using four sample lots from natural populations of the Yangtze River. The mtCR DNA sequences of approximately 920 base pairs were obtained. A total of 223 nucleotide positions were polymorphic, and these defined 39 haplotypes. Of the 39 haplotypes, 37 (90%) were not shared, and among the populations as a whole there was little sharing of haplotypes. The average haplotype diversity (0.958) and the average nucleotide diversity (0.052) indicated a higher level of genetic diversity of Chinese sucker through the river. Analysis of molecular variation (AMOVA) of data revealed significant partitioning of variance (P<0.001) among populations (60.29%), and within populations (39.71%). The topology according to the neighbor joining and maximum parsimony methods showed mosaic composition of the 39 haplotypes, suggesting that the populations wore not completely divergent. The pairwise F statistic values, however, indicated that the population structuring existed to some extent among the geographic populations. There was a positive relationship between the aquatic distance and the genetic distance (Fst) among the populations (P<0.05). Based on our data, it is suggested that genetic drift, gene flow, and stochastic events are the possible factors influencing the population structure and genetic variation.

Genetic heterogeneity analysis and RAPD marker detection among four forms of Atrina pectinata Linnaeus

Pen shell (Atrina pectinata Linnaeus) can be distinguished into four forms based on the morphololgic characteristics. Genetic similarity, and heterogeneity were analyzed among the four forms by random amplified polymorphic DNA (RAPD) technique using 24 10-nucleotide-long primers. Of these primers, 22 pruners produced well-identifiable RAPD band patterns. Significant differences in RAPD band patterns were revealed among the four forms. A total of 198 polymorphic fragments were scored from 22 pruners. and they are specific for one form, shared by two or three forms. Several pruners, such as S451, S453 S463 S464, S470. S473 and S474, produced abundant band patterns and provided sufficient information for reliable discrimination of the four forms. The average genetic distances and phylogenetic relationships were calculated and analyzed according to the distinguishable fragments. The data indicate that pen shells of form G and form Y are similar not only among individuals within the same form, but also between individuals from the two forms, and that shells of form T and form S are highly divergent. The constructed phylogenetic free matches the average genetic distances. Three clusters were clearly distinguishable, in which two were corresponding to form S and form T respectively and one included forms G and Y. This Study will be benefit to further studies oil the taxonomy and selective breeding of Pinnid species. It is suggested that the four forms of pen shell should be categorized to at least two species taxonomically.

Design and Verification of the Programming Circuit in an Application-Specific FPGA

In this paper we present a methodology and its implementation for the design and verification of programming circuit used in a family of application-specific FPGAs that share a common architecture. Each member of the family is different either in the types of functional blocks contained or in the number of blocks of each type. The parametrized design methodology is presented here to achieve this goal. Even though our focus is on the programming circuitry that provides the interface between the FPGA core circuit and the external programming hardware, the parametrized design method can be generalized to the design of entire chip for all members in the FPGA family. The method presented here covers the generation of the design RTL files and the support files for synthesis, place-and-route layout and simulations. The proposed method is proven to work smoothly within the complete chip design methodology. We will describe the implementation of this method to the design of the programming circuit in details including the design flow from the behavioral-level design to the final layout as well as the verification. Different package options and different programming modes are included in the description of the design. The circuit design implementation is based on SMIC 0.13-micron CMOS technology.